Methods and compositions for modulating alpha-1-antitrypsin expression

ABSTRACT

Disclosed herein are methods for decreasing A1AT mRNA and protein expression and treating, ameliorating, preventing, slowing progression, or stopping progression of fibrosis. Disclosed herein are methods for decreasing A1AT mRNA and protein expression and treating, ameliorating, preventing, slowing progression, or stopping progression of liver disease, such as, A1ATD associated liver disease, and pulmonary disease, such as, A1ATD associated pulmonary disease in an individual in need thereof. Methods for inhibiting A1AT mRNA and protein expression can also be used as a prophylactic treatment to prevent individuals at risk for developing a liver disease, such as, A1ATD associated liver disease and pulmonary disease, such as, A1ATD associated pulmonary disease.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0163USC4SEQ_ST25.txt created Oct. 13, 2020, which is 108 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD

Provided are methods and compositions for reducing expression of alpha-1antitrypsin (A1AT) mRNA and protein in an animal. Such methods andcompositions are useful for treating, ameliorating, preventing, slowingprogression, or stopping progression of diseases, disorders, andconditions associated with alpha-1 antitrypsin deficiency (A1ATD). Suchdiseases, disorders, and conditions associated with A1ATD include liverdiseases, such as alpha-1 antitrypsin deficiency (A1ATD) associatedliver disease, and pulmonary diseases, such as A1ATD pulmonary disease.

BACKGROUND

Alpha-1 antitrypsin (also known as α₁-antitrypsin or A1AT) is a 52 kDserpin glycoprotein produced in hepatocytes and in smaller quantities inphagocytes and lung epithelial cells (Gettins, P. G. Chem. Rev. 2002.102: 4751-4804). A1AT is a protease inhibitor.

A1AT deficiency (A1ATD) is a genetic disorder associated with thedevelopment of liver and lung disease (Bals, R. Best Pract. Res. Clin.Gastroenterol. 2010. 24: 629-633). A1AT deficiency is caused byhomozygosity for the A1AT mutant Z gene and occurs in 1 in 2,000 birthsin many North American and European populations (Teckman, J. H. Semin.Liver Dis. 2007. 27: 274-281). Additionally, recent studies havesuggested that the PiZ heterozygous state may be associated withincreased severity and worse outcome in liver disease of knownetiologies, such as HCV, alcoholic liver disease (ALD) or NAFLD (RegevA. J. Pediatr. Gastroenterol. Nutr. 2006. 43: S30-S35). In the mostcommon genetic deficiency (PiZ, resulting from a mutation of glutamateto lysine at position 342 of the gene), there is accumulation of A1AT inthe liver as a result of polymer formation (Stockley, R. A. Expert Opin.Emerg. Drugs. 2010. 15: 685-694). The abnormal mutant proteinaccumulates within the endoplasmic reticulum of hepatocytes asintrahepatocytic globules. The result of this intracellular accumulationin homozygous ZZ individuals is an increased risk of chronic liverdisease and hepatocellular carcinoma (Perlmutter, D. H. et al.,Hepatology. 2007. 45: 1313-1323; Teckman, J. H. et al., Curr.Gastroenterol. Rep. 2006. 8: 14-20; Eriksson, S. et al., Am. J. Respir.Crit. Care Med. 2003. 168: 856-869). There are no specific treatmentsfor A1ATD associated liver disease, other than liver transplantation.

A1AT deficiency also predisposes individuals to the development ofchronic obstructive pulmonary disease (COPD) and emphysema. In theabsence of wild-type A1AT, neutrophil elastase is free to break downelastin, which contributes to the elasticity of the lungs, resulting inrespiratory disorders (DeMeo, D. L. and Silverman, E. K. Thorax. 2004.59: 259-264). Also, in such patients, there is an excess of mutant A1ATpolymers, which are bound to the alveolar wall. This enables thepolymers to act as a chronic stimulus for neutrophil influx in the lungsof A1AT deficient individuals (Mahadeva, R. et al., Am. J. Pathobiol.2005. 166: 377-387).

Described herein are compositions and methods for modulating A1ATexpression. The compounds and treatment methods described herein providesignificant advantages over the treatments options currently availablefor A1AT-related disorders.

SUMMARY

Provided herein are compounds that modulate expression of A1AT mRNA andprotein.

Certain embodiments describe compounds. In certain embodiments, thecompound is an antisense compound. In certain embodiments, the compoundcomprises an oligonucleotide. In certain embodiments, theoligonucleotide is an antisense oligonucleotide. In certain embodiments,the oligonucleotide is a modified oligonucleotide. In certainembodiments, the oligonucleotide is a modified antisenseoligonucleotide. In certain embodiments, the compound comprises amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8 contiguousnucleobases of a sequence set out in the sequence listing as one of SEQID NOs: 20-41. Certain embodiments describe a composition comprising acompound. In certain embodiments, the composition comprises a compoundaccording to any of the embodiments as described herein or a saltthereof and a pharmaceutically acceptable carrier or diluent.

In certain embodiments, provided are compounds and compositionsaccording to any of the embodiments as described herein for use intherapy.

Provided herein are methods for modulating expression of A1AT mRNA andprotein. In certain embodiments, A1AT specific inhibitors modulateexpression of A1AT mRNA and protein. In certain embodiments, A1ATspecific inhibitors are nucleic acids, proteins, or small molecules.

In certain embodiments, modulation can occur in a cell or tissue. Incertain embodiments, the cell or tissue is in an animal. In certainembodiments, the animal is a human. In certain embodiments, A1AT mRNAlevels are reduced. In certain embodiments, A1AT protein levels arereduced. In certain embodiments, A1AT mRNA and protein levels arereduced. In certain embodiments, mutant A1AT mRNA and protein levels arereduced. Such reduction can occur in a time-dependent manner or in adose-dependent manner.

Also provided are methods useful for treating, ameliorating, preventing,slowing progression, or stopping progression of diseases, disorders, andconditions associated with A1ATD. In certain embodiments, such diseases,disorders, and conditions associated with A1ATD include liver diseases,such as alpha-1 antitrypsin deficiency (A1ATD) associated liver disease,viral liver disease, and nonalcoholic steatohepatitis (NASH). In certainembodiments, A1ATD exacerbates underlying liver diseases. In certainembodiments, diseases, disorders, and conditions associated with A1ATDinclude pulmonary diseases, such as alpha-1 antitrypsin deficiency(A1ATD) associated pulmonary disease, chronic obstructive pulmonarydisease (COPD), and emphysema. In certain embodiments, A1ATD exacerbatesunderlying pulmonary diseases.

Diseases, disorders, and conditions associated with A1ATD can have oneor more risk factors, causes, or outcomes in common. Certain riskfactors and causes for development of diseases, disorders, andconditions associated with A1ATD include genetic predisposition toalpha-1 antitrypsin deficiency. In certain embodiments, a defect in anindividual's genetic code for alpha-1 antitrypsin (A1AT) is responsiblefor diseases, disorders, and conditions associated with A1ATD, such as,liver diseases and pulmonary diseases. In certain embodiments, geneticmutations lead to expression of mutant A1AT. In certain embodiments,mutant A1AT forms aggregates which are retained in the liver, causingliver dysfunction or hepatic toxicity. Certain outcomes associated withliver disease, including A1ATD associated liver disease, includeabdominal swelling or pain, bruising easily, dark urine, light coloredstools, itching all over the body, vomiting blood, passing bloody orblack stools, jaundice, lack of normal weight and height gain inchildren, liver cirrhosis, and death. In certain embodiments, mutantA1AT forms aggregates which are retained in the lung, causing pulmonarydysfunction or pulmonary disease. Certain outcomes associated withpulmonary disease, including A1ATD associated pulmonary disease, includechronic cough, fatigue, respiratory infections, shortness of breath(dyspnea), wheezing, pulmonary hypertension, heart disease, and death.

In certain embodiments, methods of treatment include administering anA1AT specific inhibitor to an individual in need thereof. In certainembodiments, the A1AT specific inhibitor is a nucleic acid. In certainembodiments, the nucleic acid is an antisense compound. In certainembodiments, the antisense compound is a modified oligonucleotide. Incertain embodiments, the modified oligonucleotide reduces A1AT proteinlevels, which alleviates hepatic toxicity induced by protein aggregates.In certain embodiments, the modified oligonucleotide reduces A1ATprotein in the liver. In certain embodiments, the modifiedoligonucleotide reduces A1AT protein levels, which alleviates pulmonarydysfunction induced by protein aggregates. In certain embodiments, themodified oligonucleotide reduces A1AT protein in the lung. In certainembodiments, the A1AT protein is mutant A1AT protein.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Additionally, as used herein, the use of “and” means “and/or” unlessstated otherwise. Furthermore, the use of the term “including” as wellas other forms, such as “includes” and “included”, is not limiting.Also, terms such as “element” or “component” encompass both elements andcomponents comprising one unit and elements and components that comprisemore than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this disclosure,including, but not limited to, patents, patent applications, publishedpatent applications, articles, books, treatises, and GENBANK AccessionNumbers and associated sequence information obtainable through databasessuch as National Center for Biotechnology Information (NCBI) and otherdata referred to throughout in the disclosure herein are herebyexpressly incorporated by reference for the portions of the documentdiscussed herein, as well as in their entirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to anO-methoxy-ethyl modification of the T position of a furanosyl ring. A2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means anucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituentat the 2′-position of the furanosyl ring other than H or OH. In certainembodiments, 2′ substituted nucleosides include nucleosides withbicyclic sugar modifications.

“3′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 3′-most nucleotide of a particular antisensecompound.

“5′ target site” refers to the nucleotide of a target nucleic acid whichis complementary to the 5′-most nucleotide of a particular antisensecompound.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“A1AT nucleic acid” or “alpha-1 antitrypsin nucleic acid” means anynucleic acid encoding A1AT. For example, in certain embodiments, a A1ATnucleic acid includes a DNA sequence encoding A1AT, an RNA sequencetranscribed from DNA encoding A1 AT (including genomic DNA comprisingintrons and exons), and an mRNA sequence encoding A1AT. “A1AT mRNA”means an mRNA encoding an A1AT protein.

“A1AT specific inhibitor” refers to any agent capable of specificallyinhibiting the expression of A1AT mRNA and/or A1 AT protein at themolecular level. For example, A1AT specific inhibitors include nucleicacids (including antisense compounds), peptides, antibodies, smallmolecules, and other agents capable of inhibiting the expression of A1AT mRNA and/or A1AT protein.

“A1ATD” or “alpha-1 antitrypsin deficiency” means lack of sufficientA1AT necessary for normal function. A1ATD may occur due to expression ofmutant A1 AT, which is characterized by abnormal folding of the protein,which may cause A1AT to aggregate in a patient's liver, thus, allowingonly small amounts of A1AT to be released into the blood.

“A1ATD associated liver disease” or “alpha-1 antitrypsin deficiencyassociated liver disease” means liver dysfunction and/or hepatictoxicity associated with alpha-1 antitrypsin deficiency. Symptoms andoutcomes of A1ATD associated liver disease include abdominal swelling orpain, bruising easily, dark urine, light colored stools, itching allover his body, vomiting blood, passing bloody or black stools, jaundice,lack of normal weight and height gain in children, liver cirrhosis, anddeath. Although not limited by mechanism, it is theorized that A1ATD iscaused by a mutant form of A1AT which forms protein aggregates that areretained in a patient's liver. The presence of such aggregatesinterferes with proper function of the liver resulting in liverdysfunction and hepatic toxicity. Examples of A1ATD associated liverdiseases include, but are not limited to, cirrhosis and liver cancer.

“A1ATD associated pulmonary disease” or “alpha-1 antitrypsin deficiencyassociated pulmonary disease” means pulmonary dysfunction associatedwith alpha-1 antitrypsin deficiency. Symptoms and outcomes of A1ATDassociated pulmonary disease include chronic cough, fatigue, respiratoryinfections, shortness of breath (dyspnea), wheezing, pulmonaryhypertension, heart disease, and death. Although not limited bymechanism, it is theorized that A1ATD is caused by a mutant form of A1ATwhich forms protein aggregates that are retained in a patient's lungs.The presence of such aggregates interferes with proper function of thelungs resulting in lung dysfunction. Examples of A1ATD associatedpulmonary diseases include, but are not limited to, chronic obstructivepulmonary diseases (COPD) such as chronic bronchitis or emphysema.

“About” means within ±7% of a value. For example, if it is stated, “thecompounds affected at least about 70% inhibition of A1AT”, it is impliedthat the A1AT levels are inhibited within a range of 63% and 77%.

“Active pharmaceutical agent” means the substance or substances in apharmaceutical composition that provide a therapeutic benefit whenadministered to an individual. For example, in certain embodiments anantisense oligonucleotide targeted to A1AT is an active pharmaceuticalagent.

“Active target region” or “target region” means a region to which one ormore active antisense compounds is targeted. “Active antisensecompounds” means antisense compounds that reduce target nucleic acidlevels or protein levels.

“Administered concomitantly” refers to the co-administration of twoagents in any manner in which the pharmacological effects of both aremanifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects of both agents need not manifestthemselves at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Administering” means providing a pharmaceutical agent to an individual,and includes, but is not limited to administering by a medicalprofessional and self-administering.

“Amelioration” or “ameliorate” or “ameliorating” refers to a lesseningof at least one indicator, sign, or symptom of an associated disease,disorder, or condition. The severity of indicators may be determined bysubjective or objective measures, which are known to those skilled inthe art.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antibody” refers to a molecule characterized by reacting specificallywith an antigen in some way, where the antibody and the antigen are eachdefined in terms of the other. Antibody may refer to a complete antibodymolecule or any fragment or region thereof, such as the heavy chain, thelight chain, Fab region, and Fc region.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding. Examples of antisense compounds include, but are not limitedto, single-stranded and double-stranded compounds, such as, antisenseoligonucleotides, siRNAs and shRNAs.

“Antisense inhibition” means reduction of target nucleic acid levels ortarget protein levels in the presence of an antisense compoundcomplementary to a target nucleic acid compared to target nucleic acidlevels or target protein levels in the absence of the antisensecompound.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding region or segment of a target nucleic acid.

“Antisense mechanisms” are all those mechanisms involving hybridizationof a compound with target nucleic acid, wherein the outcome or effect ofthe hybridization is either target degradation or target occupancy withconcomitant stalling of the cellular machinery involving, for example,transcription or splicing.

“Base complementarity” refers to the capacity for the precise basepairing of nucleobases of an antisense oligonucleotide withcorresponding nucleobases in a target nucleic acid (i.e.,hybridization), and is mediated by Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen binding between corresponding nucleobases. “Bicyclicsugar moiety” means a modified sugar moiety comprising a 4 to 7 memberedring (including but not limited to a furanosyl) comprising a bridgeconnecting two atoms of the 4 to 7 membered ring to form a second ring,resulting in a bicyclic structure. In certain embodiments, the 4 to 7membered ring is a sugar ring. In certain embodiments the 4 to 7membered ring is a furanosyl. In certain such embodiments, the bridgeconnects the 2′-carbon and the 4′-carbon of the furanosyl.

“Bicyclic nucleic acid” or “BNA” or “BNA nucleosides” means nucleic acidmonomers having a bridge connecting two carbon atoms between the 4′ and2′position of the nucleoside sugar unit, thereby forming a bicyclicsugar. Examples of such bicyclic sugar include, but are not limited toA) α-L-Methyleneoxy (4′-CH₂—O-2′) LNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) LNA, (C) Ethyleneoxy (4′-(CH₂)₂—O-2′) LNA, (D) Aminooxy(4′-CH₂—O—N(R)-2′) LNA and (E) Oxyamino (4′-CH₂—N(R)—O-2′) LNA, asdepicted below.

As used herein, LNA compounds include, but are not limited to, compoundshaving at least one bridge between the 4′ and the 2′ position of thesugar wherein each of the bridges independently comprises 1 or from 2 to4 linked groups independently selected from —[C(R₁)(R₂)]_(n)—,—C(R₁)═C(R₂)—, —C(R₁)═N—, —C(═NR₁)—, —C(═O)—, —C(═S)—, —O—, —Si(R₁)₂—,—S(═O)_(x)— and —N(R₁)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4;each R₁ and R₂ is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, a heterocycle radical, a substitutedheterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclicradical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁),or sulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkyl or a protecting group.

Examples of 4′-2′ bridging groups encompassed within the definition ofLNA include, but are not limited to one of formulae: —[C(R₁)(R₂)]_(n)—,—[C(R₁)(R₂)]_(n)—O—, —C(R₁R₂)—N(R₁)—O— or —C(R₁R₂)—O—N(R₁)—.Furthermore, other bridging groups encompassed with the definition ofLNA are 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′,4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R₁)-2′ and 4′-CH₂—N(R₁)—O-2′- bridges,wherein each R₁ and R₂ is, independently, H, a protecting group orC₁-C₁₂ alkyl.

Also included within the definition of LNA are LNAs in which the2′-hydroxyl group of the ribosyl sugar ring is connected to the 4′carbon atom of the sugar ring, thereby forming a methyleneoxy(4′-CH₂—O-2′) bridge to form the bicyclic sugar moiety. The bridge canalso be a methylene (—CH₂—) group connecting the 2′ oxygen atom and the4′ carbon atom, for which the term methyleneoxy (4′-CH₂—O-2′) LNA isused. Furthermore; in the case of the bicyclic sugar moiety having anethylene bridging group in this position, the term ethyleneoxy(4′-CH₂CH₂—O-2′) LNA is used, α-L-methyleneoxy (4′-CH₂—O-2′), an isomerof methyleneoxy (4′-CH₂—O-2′) LNA is also encompassed within thedefinition of LNA, as used herein.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“cEt” or “constrained ethyl” means a bicyclic nucleoside having a sugarmoiety comprising a bridge connecting the 4′-carbon and the 2′-carbon,wherein the bridge has the formula: 4′-CH(CH₃)—O-2′.

“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleosidecomprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′-O-methoxyethylnucleotides is chemically distinct from a region having nucleotideswithout 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions.

“Co-administration” means administration of two or more pharmaceuticalagents to an individual. The two or more pharmaceutical agents may be ina single pharmaceutical composition, or may be in separatepharmaceutical compositions. Each of the two or more pharmaceuticalagents may be administered through the same or different routes ofadministration. Co-administration encompasses parallel or sequentialadministration.

“Comprise,” “comprises” and “comprising” will be understood to imply theinclusion of a stated step or element or group of steps or elements butnot the exclusion of any other step or element or group of steps orelements. “Complementarity” means the capacity for base pairing betweennucleobases of a first nucleic acid and a second nucleic acid.“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′position of the sugar portion of the nucleotide. Deoxyribonucleotidesmay be modified with any of a variety of substituents.

“Designing” or “Designed to” refer to the process of designing anoligomeric compound that specifically hybridizes with a selected nucleicacid molecule.

“Downstream” refers to the relative direction toward the 3′ end orC-terminal end of a nucleic acid.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition may be aliquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose may be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections may be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses may be stated as theamount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” means the amount of active pharmaceutical agentsufficient to effectuate a desired physiological outcome in anindividual in need of the agent. The effective amount may vary amongindividuals depending on the health and physical condition of theindividual to be treated, the taxonomic group of the individuals to betreated, the formulation of the composition, assessment of theindividual's medical condition, and other relevant factors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's codedinformation is converted into structures present and operating in acell. Such structures include, but are not limited to the products oftranscription and translation.

“Fibrosis” refers to the formation of fibrous tissue. Excess fibrosis inan organ or tissue can lead to a thickening of the affected area andscar formation. Fibrosis can lead to organ or tissue damage and adecrease in the function of the organ or tissue. Examples of fibrosisinclude, but is not limited to, cirrhosis (fibrosis of the liver) andpulmonary fibrosis (fibrosis of the lung).

“Fully complementary” or “100% complementary” means each nucleobase of afirst nucleic acid has a complementary nucleobase in a second nucleicacid. In certain embodiments, a first nucleic acid is an antisensecompound and a target nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region may be referred to as a “gap” and theexternal regions may be referred to as the “wings.”

“Gap-widened” means a chimeric antisense compound having a gap segmentof 12 or more contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from one to sixnucleosides.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude an antisense compound and a target nucleic acid.

“Identifying an animal at risk for developing A1ATD associated liverdisease” means identifying an animal having been diagnosed with A1ATDassociated liver disease or identifying an animal predisposed to developA1ATD associated liver disease. Individuals predisposed to develop anA1ATD associated liver disease include those having one or more riskfactors for A1ATD associated liver disease, including, having a personalor family history of A1ATD or A1ATD associated liver disease. Suchidentification may be accomplished by any method including evaluating anindividual's medical history and standard clinical tests or assessments,such as genetic testing.

“Identifying an animal at risk for developing A1ATD associated pulmonarydisease” means identifying an animal having been diagnosed with A1ATDassociated pulmonary disease or identifying an animal predisposed todevelop A1ATD associated pulmonary disease. Individuals predisposed todevelop an A1ATD associated pulmonary disease include those having oneor more risk factors for A1ATD associated pulmonary disease, including,having a personal or family history of A1ATD or A1ATD associatedpulmonary disease. Such identification may be accomplished by any methodincluding evaluating an individual's medical history and standardclinical tests or assessments, such as genetic testing.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease”,upregulate”, “downregulate”, or the like, generally denote quantitativedifferences between two states.

“Inhibiting A1AT” means reducing expression of A1AT mRNA and/or proteinlevels in the presence of an A1AT specific inhibitor, including an A1 ATantisense oligonucleotide, as compared to expression of A1AT mRNA and/orprotein levels in the absence of an A1AT specific inhibitor, such as anA1AT antisense oligonucleotide.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“ISIS number” refers to an A1AT specific inhibitor that is a modifiedantisense oligonucleotide having the nucleobase sequence specified bythe associated SEQ ID NO and the chemistry and motif associated in therelated example section. For example, “ISIS 487660” means an A1ATspecific inhibitor that is a modified antisense oligonucleotide havingthe nucleobase sequence (from 5′ to 3′) “CCAGCTCAACCCTTCTTTAA”,incorporated herein as SEQ ID NO: 38, a 5-10-5 MOE gapmer, wherein eachinternucleoside linkage is a phosphorothioate internucleoside linkageand each cytosine is a 5-methylcytosine, and each of nucleosides 1-5 and16-20 comprise a 2′-O-methoxyethyl sugar moiety. For example, “ISIS496407” means an A1AT specific inhibitor that is a modified antisenseoligonucleotide having the nucleobase sequence (from 5′ to 3′)“CTTCTTTAATGTCATCCAGG”, incorporated herein as SEQ ID NO: 29, a 5-10-5MOE gapmer, wherein each internucleoside linkage is a phosphorothioateinternucleoside linkage and each cytosine is a 5-methylcytosine, andeach of nucleosides 1-5 and 16-20 comprise a 2′-O-methoxyethyl sugarmoiety.

“Linked deoxynucleoside” means a nucleic acid base (A, G, C, T, U)substituted by deoxyribose linked by a phosphate ester to form anucleotide.

“Linked nucleosides” means adjacent nucleosides which are bondedtogether.

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of base pairing withthe corresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond (i.e., aphosphodiester internucleoside bond).

“Modified nucleobase” refers to any nucleobase other than adenine,cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase”means the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, amodified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, or modifiednucleobase. A “modified nucleoside” means a nucleoside having,independently, a modified sugar moiety or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising amodified internucleoside linkage, a modified sugar, or a modifiednucleobase.

“Modified sugar” refers to a substitution or change from a naturalsugar.

“Motif” means the pattern of chemically distinct regions in an antisensecompound.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Non-complementary nucleobase” refers to a pair of nucleobases that donot form hydrogen bonds with one another or otherwise supporthybridization.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids(DNA), single-stranded nucleic acids, double-stranded nucleic acids,small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of base pairing with abase of another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable ofbase pairing with another nucleobase. For example, in DNA, adenine (A)is complementary to thymine (T). For example, in RNA, adenine (A) iscomplementary to uracil (U). In certain embodiments, complementarynucleobase refers to a nucleobase of an antisense compound that iscapable of base pairing with a nucleobase of its target nucleic acid.For example, if a nucleobase at a certain position of an antisensecompound is capable of hydrogen bonding with a nucleobase at a certainposition of a target nucleic acid, then the position of hydrogen bondingbetween the oligonucleotide and the target nucleic acid is considered tobe complementary at that nucleobase pair.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugaror the sugar and the base and not necessarily the linkage at one or morepositions of an oligomeric compound such as for example nucleosidemimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl,bicyclo, or tricyclo sugar mimetics, e.g., non furanose sugar units.Nucleotide mimetic includes those structures used to replace thenucleoside and the linkage at one or more positions of an oligomericcompound such as for example peptide nucleic acids or morpholinos(morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiesterlinkage). Sugar surrogate overlaps with the slightly broader termnucleoside mimetic but is intended to indicate replacement of the sugarunit (furanose ring) only. The tetrahydropyranyl rings provided hereinare illustrative of an example of a sugar surrogate wherein the furanosesugar group has been replaced with a tetrahydropyranyl ring system.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Oligomeric compound” or “oligomer” means a polymer of linked monomericsubunits which is capable of hybridizing to at least a region of anucleic acid molecule.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g., intrathecal orintracerebroventricular administration.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Peptide refers to polypeptides and proteins.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual. For example, a pharmaceuticalcomposition may comprise one or more active pharmaceutical agents and asterile aqueous solution.

“Pharmaceutically acceptable derivative” encompasses pharmaceuticallyacceptable salts, conjugates, prodrugs or isomers of the compoundsdescribed herein.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage (P═S) is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e., linked)nucleobases of a nucleic acid. In certain embodiments, a portion is adefined number of contiguous nucleobases of a target nucleic acid. Incertain embodiments, a portion is a defined number of contiguousnucleobases of an antisense compound.

“Prevent” or “preventing” refers to delaying or forestalling the onsetor development of a disease, disorder, or condition for a period of timefrom minutes to indefinitely. Prevent also means reducing risk ofdeveloping a disease, disorder, or condition.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form within the body or cells thereof bythe action of endogenous enzymes or other chemicals or conditions.

“Pulmonary administration” means administration topical to the surfaceof the respiratory tract. Pulmonary administration includesnebulization, inhalation, or insufflation of powders or aerosols, bymouth and/or nose.

“Region” is defined as a portion of the target nucleic acid having atleast one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ positionof the sugar portion of the nucleotide. Ribonucleotides may be modifiedwith any of a variety of substituents.

“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid.

“Sites,” as used herein, are defined as unique nucleobase positionswithin a target nucleic acid. “Side effects” means physiologicalresponses attributable to a treatment other than the desired effects. Incertain embodiments, side effects include injection site reactions,liver function test abnormalities, renal function abnormalities, livertoxicity, renal toxicity, central nervous system abnormalities,myopathies, and malaise. For example, increased aminotransferase levelsin serum may indicate liver toxicity or liver function abnormality. Forexample, increased bilirubin may indicate liver toxicity or liverfunction abnormality.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity between an antisenseoligonucleotide and a target nucleic acid to induce a desired effect,while exhibiting minimal or no effects on non-target nucleic acids underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays and therapeutictreatments.

“Subject” means a human or non-human animal selected for treatment ortherapy.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” allrefer to a nucleic acid capable of being targeted by antisensecompounds.

“Target region” means a portion of a target nucleic acid to which one ormore antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceuticalagent that provides a therapeutic benefit to an individual.

“Treat” or “treating” refers to administering a pharmaceuticalcomposition to effect an alteration or improvement of a disease,disorder, or condition.

“Unmodified” nucleobases mean the purine bases adenine (A) and guanine(G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

Upstream” refers to the relative direction toward the 5′ end orN-terminal end of a nucleic acid.

CERTAIN EMBODIMENTS

Certain embodiments provide methods for decreasing A1AT mRNA and proteinexpression.

Certain embodiments provide methods for the treatment, amelioration, orprevention of diseases, disorders, and conditions associated with A1ATin an individual in need thereof. Also contemplated are methods for thepreparation of a medicament for the treatment, amelioration, orprevention of a disease, disorder, or condition associated with A1 AT.In certain embodiments, A1AT associated diseases, disorders, andconditions include liver disease, such as, A1ATD associated liverdisease. In certain embodiments, A1AT associated diseases, disorders,and conditions include pulmonary diseases, such as, A1ATD associatedpulmonary disease, COPD, or emphysema.

Such diseases, disorders, and conditions may have one or more riskfactors, causes, or outcomes in common. Certain risk factors and causesfor development of diseases, disorders, and conditions associated withA1ATD include genetic predisposition to alpha-1 antitrypsin deficiency.In certain embodiments, a defect in an individual's genetic code foralpha-1 antitrypsin (A1AT) is responsible for diseases, disorders, andconditions associated with A1ATD, such as, liver diseases and pulmonarydiseases. In certain embodiments, genetic mutations lead to expressionof mutant A1AT. In certain embodiments, mutant A1AT forms aggregateswhich are retained in the liver, causing liver dysfunction or hepatictoxicity. Certain outcomes associated with liver disease, includingA1ATD associated liver disease, include abdominal swelling or pain,bruising easily, dark urine, light colored stools, itching all over thebody, vomiting blood, passing bloody or black stools, jaundice, lack ofnormal weight and height gain in children, liver cirrhosis, and death.In certain embodiments, mutant A1AT forms aggregates which are retainedin the lung, causing pulmonary dysfunction or pulmonary disease. Certainoutcomes associated with pulmonary disease, including A1ATD associatedpulmonary disease, include chronic cough, fatigue, respiratoryinfections, shortness of breath (dyspnea), wheezing, pulmonaryhypertension, heart disease, and death.

Certain embodiments provide for the use of an A1AT specific inhibitorfor treating, ameliorating, preventing, slowing progression, or stoppingprogression of an A1AT associated disease. In certain embodiments, A1ATspecific inhibitors are nucleic acids (including antisense compounds),peptides, antibodies, small molecules, and other agents capable ofinhibiting the expression of A1AT mRNA and/or A1AT protein.

In certain embodiments, methods of treatment include administering anA1AT specific inhibitor to an individual in need thereof. In certainembodiments, A1AT specific inhibitors are antisense compounds. Incertain embodiments, the antisense compound is a modifiedoligonucleotide targeting A1AT.

Certain embodiments provide a method of reducing A1AT in an animalcomprising administering to the animal a modified oligonucleotidetargeting an A1AT nucleic acid sequence as shown in SEQ ID NO: 1. Incertain embodiments, the modified oligonucleotide targeting A1ATconsists of 12 to 30 linked nucleosides and is at least 90%complementary to the A1AT nucleic acid.

Certain embodiments provide a method of treating, ameliorating and/orpreventing an A1ATD associated liver disease in an animal at risk forthe A1ATD associated liver disease comprising: (a) identifying theanimal at risk for developing the A1ATD associated liver disease; and(b) administering to the at risk animal a therapeutically effectiveamount of a modified oligonucleotide consisting of 12 to 30 linkednucleosides, wherein the modified oligonucleotide is at least 90%complementary to an A1AT nucleic acid.

Certain embodiments provide a method of treating, ameliorating and/orpreventing an A1ATD associated pulmonary disease in an animal at riskfor the A1ATD associated pulmonary disease comprising: (a) identifyingthe animal at risk for developing the A1ATD associated pulmonarydisease; and (b) administering to the at risk animal a therapeuticallyeffective amount of a modified oligonucleotide consisting of 12 to 30linked nucleosides, wherein the modified oligonucleotide is at least 90%complementary to an A1 AT nucleic acid.

Certain embodiments provide a method of halting progression of an A1ATDassociated liver disease comprising: (a) identifying an animal with theA1ATD associated liver disease; and (b) administering to the animal atherapeutically effective amount of a modified oligonucleotideconsisting of 12 to 30 linked nucleosides, wherein the modifiedoligonucleotide is at least 90% complementary to an A1AT nucleic acid.

Certain embodiments provide a method of lowering the risk for developingan A1ATD associated liver disease comprising: (a) identifying an animalat risk for developing A1ATD associated liver disease; and (b)administering to the at risk animal a therapeutically effective amountof a modified oligonucleotide consisting of 12 to 30 linked nucleosides,wherein the modified oligonucleotide is at least 90% complementary to anA1AT nucleic acid.

Certain embodiments provide a method of reducing fibrosis in an animalcomprising: (a) identifying the animal at risk for developing fibrosis;and (b) administering to the at risk animal a therapeutically effectiveamount of a modified oligonucleotide consisting of 12 to 30 linkednucleosides, wherein the modified oligonucleotide is at least 90%complementary to an A1AT nucleic acid.

Certain embodiments provide a method of preventing organ damage,decreasing organ damage and/or improving organ function in an animalcomprising: (a) identifying the animal at risk for organ damage ordecrease organ function; and (b) administering to the at risk animal atherapeutically effective amount of a modified oligonucleotideconsisting of 12 to 30 linked nucleosides, wherein the modifiedoligonucleotide is at least 90% complementary to an A1AT nucleic acid.

In certain embodiments, accumulation of A1AT aggregates is forestalled,prevented or delayed in an animal by the methods provided herein. Incertain embodiments, the accumulation of A1AT aggregates is in a lung,liver or other organ or tissue.

In certain embodiments, the methods provided herein decreases TIMP1,collagen type 1, collagen type IV, collagen type III, MMP13, SMA, ALTand/or AST expression.

Embodiments described herein provide the use of an A1AT specificinhibitor, as described herein, for use in treating, ameliorating,preventing, slowing progression, or stopping progression of a liverdisease, such as A1ATD associated liver disease, as described herein, bycombination therapy with an additional agent or therapy, as describedherein. Agents or therapies can be co-administered or administeredconcomitantly. In certain embodiments, A1AT specific inhibitors areantisense compounds.

Embodiments described herein provide the use of an A1AT specificinhibitor, as described herein, for use in treating, ameliorating,preventing, slowing progression, or stopping progression of a pulmonarydisease, such as A1ATD associated pulmonary disease, as describedherein, by combination therapy with an additional agent or therapy, asdescribed herein. Agents or therapies can be co-administered oradministered concomitantly. In certain embodiments, A1AT specificinhibitors are antisense compounds.

In certain embodiments, A1AT specific inhibitors are peptides orproteins (Chang, Y. P. et al., Am. J. Respir. Cell Mol. Biol. 2006. 35:540-548) or ribozymes (Zem, M. A. et al., Gene Ther. 1999. 6: 114-120),and FLEAIG peptide (US 20110280863). In certain embodiments, A1ATspecific inhibitors are antibodies (Miranda, E. et al., Hepatology.2010. 52: 1078-1088; Piotrowska, U. et al., Thyroid. 2002. 12: 563-570).

In certain embodiments, A1AT specific inhibitors are small molecules,such as, but not limited to, rapamycin (Kaushal, S. et al., Exp. Biol.Med. 2010. 235: 700-709), 4-phenylbutyric acid, which preventsmisfolding of A1AT (Burrows, J. A. et al., Proc. Natl. Acad. Sci. U.S.A.2000. 97: 1796-1801), nafamostat mesilate (Sundaram, S. et al., Thromb.Haemost. 1996. 75: 76-82), trimethylamine N-oxide (US 20110280863),1-deoxynojirimycin (Tan, A. et al., J. Biol. Chem. 1991.266:14504-14510), and D-galactosamine (Gross, V. et al., Biochim. Biophys.Acta. 1990. 1036: 143-150).

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andcomprising a nucleobase sequence comprising a portion of at least 8, atleast 10, at least 12, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, or at least contiguous nucleobasescomplementary to an equal length portion of nucleobases 1575 to 1594 ofSEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 90% complementary to SEQ ID NO: 1.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andcomprising a nucleobase sequence comprising a portion of at least 8, atleast 10, at least 12, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, or at least contiguous nucleobasescomplementary to an equal length portion of nucleobases 1564 to 1583 ofSEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 90% complementary to SEQ ID NO: 1.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andcomprising a nucleobase sequence comprising a portion of at least 8, atleast 10, at least 12, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, or at least contiguous nucleobasescomplementary to an equal length portion of nucleobases 1561 to 1597 ofSEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 90% complementary to SEQ ID NO: 1.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andcomprising a nucleobase sequence comprising a portion of at least 8, atleast 10, at least 12, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, or at least contiguous nucleobasescomplementary to an equal length portion of nucleobases 1349 to 1597 ofSEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 90% complementary to SEQ ID NO: 1.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andcomprising a nucleobase sequence comprising a portion of at least 8, atleast 10, at least 12, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, or at least contiguous nucleobasescomplementary to an equal length portion of nucleobases 459 to 513 ofSEQ ID NO: 1, wherein the nucleobase sequence of the modifiedoligonucleotide is at least 90% complementary to SEQ ID NO: 1.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 10, atleast 12, at least 14, at least 16, at least 17, at least 18, at least19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ IDNO: 38.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 10, atleast 12, at least 14, at least 16, at least 17, at least 18, at least19, or 20 contiguous nucleobases of the nucleobase sequence of SEQ IDNO: 29.

In certain embodiments, provided herein are compounds comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 8, at least 10, atleast 12, at least 14, at least 16, at least 17, at least 18, at least19, or 20 contiguous nucleobases of the nucleobase sequences of SEQ IDNO: 20-41.

In certain embodiments, the modified oligonucleotide consists of 15 to30, 18 to 24, 19 to 22, or 20 linked nucleosides.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of asingle-stranded modified oligonucleotide.

In certain embodiments, at least one internucleoside linkage of themodified oligonucleotide is a modified internucleoside linkage. Incertain embodiments, at least one modified oligonucleotide is aphosphorothioate internucleoside linkage. In certain embodiments, eachinternucleoside linkage is a phosphorothioate internucleoside linkage.

In certain embodiments, at least one nucleoside of the modifiedoligonucleotide comprises a modified nucleobase. In certain embodiments,the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide comprises at leastone modified sugar. In certain embodiments, the modified sugar is a2′-O-methoxyethyl.

In certain embodiments, the modified oligonucleotide comprises at leastone 2′-O-methoxyethyl nucleoside.

In certain embodiments, the modified sugar is a bicyclic sugar. Incertain embodiments, the bicyclic sugar comprises a 4′-CH(CH₃)—O-2′bridge.

In certain embodiments, the modified oligonucleotide comprises:

a gap segment consisting of linked deoxynucleosides;

a 5′ wing segment consisting of linked nucleosides;

a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment and wherein each nucleoside of each wing segmentcomprises a modified sugar.

In certain embodiments, the modified oligonucleotide comprises:

a gap segment consisting of ten linked deoxynucleosides;

a 5′ wing segment consisting of five linked nucleosides;

a 3′ wing segment consisting of five linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment andthe 3′ wing segment, wherein each nucleoside of each wing segmentcomprises a 2′-O-methoxyethyl sugar; wherein each internucleosidelinkage is a phosphorothioate linkage; and wherein each cytosine is a5′-methylcytosine.

In certain embodiments, the modified oligonucleotide consists of 15 to24 linked nucleosides and comprises a nucleobase sequence comprising aportion of at least 15 contiguous nucleobases complementary to an equallength portion of nucleobases 1575 to 1594 of SEQ ID NO: 1, and whereinthe nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 15 to24 linked nucleosides and comprises a nucleobase sequence comprising aportion of at least 15 contiguous nucleobases complementary to an equallength portion of nucleobases 1564 to 1583 of SEQ ID NO: 1, and whereinthe nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 15 to24 linked nucleosides and comprises a nucleobase sequence comprising aportion of at least 15 contiguous nucleobases complementary to an equallength portion of nucleobases 1561 to 1597 of SEQ ID NO: 1, and whereinthe nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 15 to24 linked nucleosides and comprises a nucleobase sequence comprising aportion of at least 15 contiguous nucleobases complementary to an equallength portion of nucleobases 1349 to 1597 of SEQ ID NO: 1, and whereinthe nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 15 to24 linked nucleosides and comprises a nucleobase sequence comprising aportion of at least 15 contiguous nucleobases complementary to an equallength portion of nucleobases 459 to 513 of SEQ ID NO: 1, and whereinthe nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 18 to24 linked nucleosides and comprises a nucleobase sequence comprising aportion of at least 18 contiguous nucleobases complementary to an equallength portion of nucleobases 1575 to 1594 of SEQ ID NO: 1, and whereinthe nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 18 to24 linked nucleosides and comprises a nucleobase sequence comprising aportion of at least 18 contiguous nucleobases complementary to an equallength portion of nucleobases 1564 to 1583 of SEQ ID NO: 1, and whereinthe nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 18 to24 linked nucleosides and comprises a nucleobase sequence comprising aportion of at least 18 contiguous nucleobases complementary to an equallength portion of nucleobases 1561 to 1597 of SEQ ID NO: 1, and whereinthe nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 18 to24 linked nucleosides and comprises a nucleobase sequence comprising aportion of at least 18 contiguous nucleobases complementary to an equallength portion of nucleobases 1349 to 1597 of SEQ ID NO: 1, and whereinthe nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO: 1.

In certain embodiments, the modified oligonucleotide consists of 18 to24 linked nucleosides and comprises a nucleobase sequence comprising aportion of at least 18 contiguous nucleobases complementary to an equallength portion of nucleobases 459 to 513 of SEQ ID NO: 1, and whereinthe nucleobase sequence of the modified oligonucleotide is 100%complementary to SEQ ID NO: 1.

In certain embodiments, provided herein are compositions comprising acompound as described herein or a salt thereof and a pharmaceuticallyacceptable carrier or diluent.

In certain embodiments, provided herein are compositions as describedherein for use in therapy.

In certain embodiments, provided herein are compositions as describedherein for use in treating, ameliorating, or preventing an A1ATdeficiency (A1ATD).

In certain embodiments, provided herein are compositions as describedherein for use in treating an individual with a genetic predispositionto an A1ATD.

In certain embodiments, the compounds and compositions described hereinare for use in treating, ameliorating, or preventing A1ATD associatedliver disease.

In certain embodiments, the compounds and compositions described hereinare for preventing or delaying A1AT aggregation in the liver.

In certain embodiments, the compounds and compositions described hereinare for use in treating, ameliorating, or preventing A1ATD associatedliver dysfunction.

In certain embodiments, the compounds and compositions described hereinare for use in treating, ameliorating, or preventing A1ATD associatedhepatic toxicity.

In certain embodiments, the compounds and compositions described hereinare for use in treating, ameliorating, or preventing A1ATD associatedpulmonary disease, COPD, and/or emphysema.

In certain embodiments, the compounds and compositions described hereinare for preventing or delaying A1AT aggregation in the lungs.

In certain embodiments, the compounds and compositions described hereinare for use in treating, ameliorating, or preventing A1ATD associatedpulmonary dysfunction.

In certain embodiments, the compounds and compositions described hereinare for use in treating, ameliorating, or preventing A1ATD associatedpulmonary toxicity.

In certain embodiments, provided herein are methods comprising,identifying an animal at risk for developing A1ATD associated liverdisease; and administering to the at risk animal a therapeuticallyeffective amount of a modified oligonucleotide consisting of 12 to 30linked nucleosides, wherein the modified oligonucleotide is at least 90%complementary to an A1AT nucleic acid.

In certain embodiments, provided herein are methods comprising,

identifying an animal at risk for developing A1ATD associated pulmonarydisease; and

administering to the at risk animal a therapeutically effective amountof a modified oligonucleotide consisting of 12 to 30 linked nucleosides,wherein the modified oligonucleotide is at least 90% complementary to anA1AT nucleic acid.

Certain embodiments describe a method of slowing or halting progressionof A1ATD associated liver disease by administering a compound orcomposition described herein. In certain embodiments, provided is amethod comprising administering a modified oligonucleotide consisting of12 to 30 linked nucleosides, wherein the modified oligonucleotide is atleast 90% complementary to an A1AT nucleic acid, and wherein theprogression of the A1ATD associated liver disease is slowed or halted.In certain embodiments, provided is a method comprising identifying ananimal having an A1ATD-associated liver disease and administering to theanimal a therapeutically effective amount of a modified oligonucleotideconsisting of 12 to 30 linked nucleosides, wherein the modifiedoligonucleotide is at least 90% complementary to an A1AT nucleic acid,and wherein the progression of the A1ATD associated liver disease isslowed or halted. In certain embodiments, the nucleobase sequence of themodified oligonucleotide is at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% complementary to an A1AT nucleic acid.

Certain embodiments describe a method of preventing or stopping ordelaying or forestalling the accumulation or onset of accumulation ofA1AT globules in the liver. In certain embodiments, the risk ofA1ATD-associated liver disease is lowered or reduced. In certainembodiments, the development or onset of A1ATD-associated liver diseaseis prevented, delayed or forestalled. In certain embodiments, providedis a method comprising administering a modified oligonucleotideconsisting of 12 to 30 linked nucleosides, wherein the modifiedoligonucleotide is at least 90% complementary to an A1AT nucleic acid,and wherein accumulation of A1AT globules in the liver is stopped,delayed or forestalled. In certain embodiments, provided is a methodcomprising administering a modified oligonucleotide consisting of 12 to30 linked nucleosides, wherein the modified oligonucleotide is at least90% complementary to an A1AT nucleic acid, and wherein the developmentor onset of A1ATD-associated liver disease is prevented, delayed orforestalled. In certain embodiments, provided is a method comprisingidentifying an animal at risk for developing A1ATD-associated liverdisease and administering to the at risk animal a therapeuticallyeffective amount of a modified oligonucleotide consisting of 12 to 30linked nucleosides, wherein the modified oligonucleotide is at least 90%complementary to an A1AT nucleic acid, and wherein the risk of A1ATDassociated liver disease is lowered or reduced. In certain embodiments,provided is a method comprising identifying an animal at risk fordeveloping A1ATD-associated liver disease and administering to the atrisk animal a therapeutically effective amount of a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides, wherein themodified oligonucleotide is at least 90% complementary to an A1ATnucleic acid, and wherein accumulation of A1AT globules or aggregates inthe liver is stopped, delayed or forestalled thereby reducing orlowering the risk of A1ATD associated liver disease. In certainembodiments, the nucleobase sequence of the modified oligonucleotide isat least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%complementary to an A1AT nucleic acid.

In certain embodiments, the modified oligonucleotide consists of 12 to30 linked nucleosides and has a nucleobase sequence comprising at least12, at least 14, at least 16, at least 17, at least 18, at least 19, orat least 20 contiguous nucleobases of the nucleobase sequences of SEQ IDNO: 20-41.

In certain embodiments, expression of A1AT mRNA is reduced.

In certain embodiments, expression of A1AT protein is reduced.

In certain embodiments, A1ATD associated liver disease is treated,ameliorated, or prevented.

In certain embodiments, A1AT aggregation in the liver is prevented ordelayed.

In certain embodiments, A1ATD associated pulmonary disease is treated,ameliorated, or prevented.

In certain embodiments, A1AT aggregation in the lung is prevented ordelayed.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound may be “antisense” to a target nucleic acid, meaningthat it is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to an A1ATnucleic acid is 12 to 30 subunits in length. In other words, suchantisense compounds are from 12 to 30 linked subunits. In otherembodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18to 24, 19 to 22, or 20 linked subunits. In certain such embodiments, theantisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a rangedefined by any two of the above values. In some embodiments theantisense compound is an antisense oligonucleotide, and the linkedsubunits are nucleosides.

In certain embodiments antisense oligonucleotides targeted to an A1ATnucleic acid may be shortened or truncated. For example, a singlesubunit may be deleted from the 5′ end (5′ truncation), or alternativelyfrom the 3′ end (3′ truncation). A shortened or truncated antisensecompound targeted to a A1AT nucleic acid may have two subunits deletedfrom the 5′ end, or alternatively may have two subunits deleted from the3′ end, of the antisense compound. Alternatively, the deletednucleosides may be dispersed throughout the antisense compound, forexample, in an antisense compound having one nucleoside deleted from the5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisensecompound, the additional subunit may be located at the 5′ or 3′ end ofthe antisense compound. When two or more additional subunits arepresent, the added subunits may be adjacent to each other, for example,in an antisense compound having two subunits added to the 5′ end (5′addition), or alternatively to the 3′ end (3′ addition), of theantisense compound. Alternatively, the added subunits may be dispersedthroughout the antisense compound, for example, in an antisense compoundhaving one subunit added to the 5′ end and one subunit added to the 3′end.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a seriesof tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Certain Antisense Compound Motifs and Mechanisms

In certain embodiments, antisense compounds have chemically modifiedsubunits arranged in patterns, or motifs, to confer to the antisensecompounds properties such as enhanced inhibitory activity, increasedbinding affinity for a target nucleic acid, or resistance to degradationby in vivo nucleases. Chimeric antisense compounds typically contain atleast one region modified so as to confer increased resistance tonuclease degradation, increased cellular uptake, increased bindingaffinity for the target nucleic acid, and/or increased inhibitoryactivity. A second region of a chimeric antisense compound may conferanother desired property e.g., serve as a substrate for the cellularendonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense activity may result from any mechanism involving thehybridization of the antisense compound (e.g., oligonucleotide) with atarget nucleic acid, wherein the hybridization ultimately results in abiological effect. In certain embodiments, the amount and/or activity ofthe target nucleic acid is modulated. In certain embodiments, the amountand/or activity of the target nucleic acid is reduced. In certainembodiments, hybridization of the antisense compound to the targetnucleic acid ultimately results in target nucleic acid degradation. Incertain embodiments, hybridization of the antisense compound to thetarget nucleic acid does not result in target nucleic acid degradation.In certain such embodiments, the presence of the antisense compoundhybridized with the target nucleic acid (occupancy) results in amodulation of antisense activity. In certain embodiments, antisensecompounds having a particular chemical motif or pattern of chemicalmodifications are particularly suited to exploit one or more mechanisms.In certain embodiments, antisense compounds function through more thanone mechanism and/or through mechanisms that have not been elucidated.Accordingly, the antisense compounds described herein are not limited byparticular mechanism.

Antisense mechanisms include, without limitation, RNase H mediatedantisense; RNAi mechanisms, which utilize the RISC pathway and include,without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancybased mechanisms. Certain antisense compounds may act through more thanone such mechanism and/or through additional mechanisms.

RNase H-Mediated Antisense

In certain embodiments, antisense activity results at least in part fromdegradation of target RNA by RNase H. RNase H is a cellular endonucleasethat cleaves the RNA strand of an RNA:DNA duplex. It is known in the artthat single-stranded antisense compounds which are “DNA-like” elicitRNase H activity in mammalian cells. Accordingly, antisense compoundscomprising at least a portion of DNA or DNA-like nucleosides mayactivate RNase H, resulting in cleavage of the target nucleic acid. Incertain embodiments, antisense compounds that utilize RNase H compriseone or more modified nucleosides. In certain embodiments, such antisensecompounds comprise at least one block of 1-8 modified nucleosides. Incertain such embodiments, the modified nucleosides do not support RNaseH activity. In certain embodiments, such antisense compounds aregapmers, as described herein. In certain such embodiments, the gap ofthe gapmer comprises DNA nucleosides. In certain such embodiments, thegap of the gapmer comprises DNA-like nucleosides. In certain suchembodiments, the gap of the gapmer comprises DNA nucleosides andDNA-like nucleosides.

Certain antisense compounds having a gapmer motif are consideredchimeric antisense compounds. In a gapmer an internal region having aplurality of nucleotides that supports RNaseH cleavage is positionedbetween external regions having a plurality of nucleotides that arechemically distinct from the nucleosides of the internal region. In thecase of an antisense oligonucleotide having a gapmer motif, the gapsegment generally serves as the substrate for endonuclease cleavage,while the wing segments comprise modified nucleosides. In certainembodiments, the regions of a gapmer are differentiated by the types ofsugar moieties comprising each distinct region. The types of sugarmoieties that are used to differentiate the regions of a gapmer may insome embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides,2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOEand 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides(such bicyclic sugar modified nucleosides may include those having aconstrained ethyl). In certain embodiments, nucleosides in the wings mayinclude several modified sugar moieties, including, for example 2′-MOEand bicyclic sugar moieties such as constrained ethyl or LNA. In certainembodiments, wings may include several modified and unmodified sugarmoieties. In certain embodiments, wings may include various combinationsof 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethylnucleosides or LNA nucleosides, and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, oralternating sugar moieties. The wing-gap-wing motif is frequentlydescribed as “X-Y-Z”, where “X” represents the length of the 5′-wing,“Y” represents the length of the gap, and “Z” represents the length ofthe 3′-wing. “X” and “Z” may comprise uniform, variant, or alternatingsugar moieties. In certain embodiments, “X” and “Y” may include one ormore 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As usedherein, a gapmer described as “X-Y-Z” has a configuration such that thegap is positioned immediately adjacent to each of the 5′-wing and the 3′wing. Thus, no intervening nucleotides exist between the 5′-wing andgap, or the gap and the 3′-wing. Any of the antisense compoundsdescribed herein can have a gapmer motif. In certain embodiments, “X”and “Z” are the same; in other embodiments they are different. Incertain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z canbe any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30 or more nucleosides.

In certain embodiments, the antisense compound has a gapmer motif inwhich the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16linked nucleosides.

In certain embodiments, the antisense oligonucleotide has a sugar motifdescribed by Formula A as follows:(J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; xis 0-2; y is 0-2; z is 0-4; g is 6-14; provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

RNAi Compounds

In certain embodiments, antisense compounds are interfering RNAcompounds (RNAi), which include double-stranded RNA compounds (alsoreferred to as short-interfering RNA or siRNA) and single-stranded RNAicompounds (or ssRNA). Such compounds work at least in part through theRISC pathway to degrade and/or sequester a target nucleic acid (thus,include microRNA/microRNA-mimic compounds). In certain embodiments,antisense compounds comprise modifications that make them particularlysuited for such mechanisms.

i. ssRNA Compounds

In certain embodiments, antisense compounds including those particularlysuited for use as single-stranded RNAi compounds (ssRNA) comprise amodified 5′-terminal end. In certain such embodiments, the 5′-terminalend comprises a modified phosphate moiety. In certain embodiments, suchmodified phosphate is stabilized (e.g., resistant todegradation/cleavage compared to unmodified 5′-phosphate). In certainembodiments, such 5′-terminal nucleosides stabilize the 5′-phosphorousmoiety. Certain modified 5′-terminal nucleosides may be found in theart, for example in WO/2011/139702.

In certain embodiments, the 5′-nucleoside of an ssRNA compound hasFormula IIe:

wherein:

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of FormulaIIe to the oligomeric compound;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄ R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇),OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl,C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

R₁₅, R₁₆, R₁₇ and R₁₈ are each, independently, H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl;

Bx₁ is a heterocyclic base moiety;

or if Bx₂ is present then Bx₂ is a heterocyclic base moiety and Bx₁ isH, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy,substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl,C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl;

or J₄ forms a bridge with one of J₅ or J₇ wherein said bridge comprisesfrom 1 to 3 linked biradical groups selected from O, S, NR₁₉,C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other twoof J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₈)(R₉)]_(n)—[(C═O)_(m)—X₁]_(j)Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl orN(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substitutedC₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protectedsubstituent groups independently selected from halogen, OJ₁, N(J₁)(J₂),═NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

wherein said oligomeric compound comprises from 8 to 40 monomericsubunits and is hybridizable to at least a portion of a target nucleicacid.

In certain embodiments, M₃ is O, CH═CH, OCH₂ or OC(H)(Bx₂). In certainembodiments, M₃ is O.

In certain embodiments, J₄, J₅, J₆ and J₇ are each H. In certainembodiments, J₄ forms a bridge with one of J₅ or J₇.

In certain embodiments, A has one of the formulas:

wherein:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substitutedC₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy. In certainembodiments, Q₁ and Q₂ are each H. In certain embodiments, Q₁ and Q₂ areeach, independently, H or halogen. In certain embodiments, Q₁ and Q₂ isH and the other of Q₁ and Q₂ is F, CH₃ or OCH₃.

In certain embodiments, T₁ has the formula:

wherein:

R_(a) and R_(c) are each, independently, protected hydroxyl, protectedthiol, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substitutedC₁-C₆ alkoxy, protected amino or substituted amino; and

R_(b) is O or S. In certain embodiments, R_(b) is O and R_(a) and R_(c)are each, independently, OCH₃, OCH₂CH₃ or CH(CH₃)₂.

In certain embodiments, G is halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃,O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃,O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁₀)(R₁₁), O(CH₂)₂—ON(R₁₀)(R₁₁),O(CH₂)₂—O(CH₂)₂—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₀)(R₁₁),OCH₂C(═O)—N(R₁₂)—(CH₂)₂—N(R₁₀)(R₁₁) orO(CH₂)₂—N(R₁₂)—C(═NR₁₃)[N(R₁₀)(R₁₁)] wherein R₁₀, R₁₁, R₁₂ and R₁₃ areeach, independently, H or C₁-C₆ alkyl. In certain embodiments, G ishalogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃,O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃,OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂. In certainembodiments, G is F, OCH₃ or O(CH₂)₂—OCH₃. In certain embodiments, G isO(CH₂)₂—OCH₃.

In certain embodiments, the 5′-terminal nucleoside has Formula IIe:

In certain embodiments, antisense compounds, including thoseparticularly suitable for ssRNA comprise one or more type of modifiedsugar moieties and/or naturally occurring sugar moieties arranged alongan oligonucleotide or region thereof in a defined pattern or sugarmodification motif. Such motifs may include any of the sugarmodifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of aregion having uniform sugar modifications. In certain such embodiments,each nucleoside of the region comprises the same RNA-like sugarmodification. In certain embodiments, each nucleoside of the region is a2′-F nucleoside. In certain embodiments, each nucleoside of the regionis a 2′-OMe nucleoside. In certain embodiments, each nucleoside of theregion is a 2′-MOE nucleoside. In certain embodiments, each nucleosideof the region is a cEt nucleoside. In certain embodiments, eachnucleoside of the region is an LNA nucleoside. In certain embodiments,the uniform region constitutes all or essentially all of theoligonucleotide. In certain embodiments, the region constitutes theentire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, oligonucleotides comprise one or more regions ofalternating sugar modifications, wherein the nucleosides alternatebetween nucleotides having a sugar modification of a first type andnucleotides having a sugar modification of a second type. In certainembodiments, nucleosides of both types are RNA-like nucleosides. Incertain embodiments the alternating nucleosides are selected from:2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, thealternating modifications are 2′-F and 2′-OMe. Such regions may becontiguous or may be interrupted by differently modified nucleosides orconjugated nucleosides.

In certain embodiments, the alternating region of alternatingmodifications each consist of a single nucleoside (i.e., the pattern is(AB)_(x)A_(y) wherein A is a nucleoside having a sugar modification of afirst type and B is a nucleoside having a sugar modification of a secondtype; x is 1-20 and y is 0 or 1). In certain embodiments, one or morealternating regions in an alternating motif includes more than a singlenucleoside of a type. For example, oligonucleotides may include one ormore regions of any of the following nucleoside motifs:

AABBAA; ABBABB; AABAAB; ABBABAABB; ABABAA; AABABAB; ABABAA;ABBAABBABABAA; BABBAABBABABAA; or ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of asecond type. In certain embodiments, A and B are each selected from2′-F, 2′-OMe, BNA, and MOE.

In certain embodiments, oligonucleotides having such an alternatingmotif also comprise a modified 5′ terminal nucleoside, such as those offormula He or He.

In certain embodiments, oligonucleotides comprise a region having a2-2-3 motif. Such regions comprises the following motif:

-(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃-

wherein: A is a first type of modified nucleoside;

B and C, are nucleosides that are differently modified than A, however,B and C may have the same or different modifications as one another;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certainembodiments, B and C are both 2′-F modified nucleosides. In certainembodiments, A is a 2′-OMe modified nucleoside and B and C are both 2′-Fmodified nucleosides.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(AB)_(x)A_(y)-(D)_(z)

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certainembodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

B is a second type of modified nucleoside;

D is a modified nucleoside comprising a modification different from thenucleoside adjacent to it. Thus, if y is 0, then D must be differentlymodified than B and if y is 1, then D must be differently modified thanA. In certain embodiments, D differs from both A and B.

X is 5-15;

Y is 0 or 1;

Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(A)_(x)-(D)_(z)

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certainembodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

D is a modified nucleoside comprising a modification different from A.

X is 11-30;

Z is 0-4.

In certain embodiments A, B, C, and D in the above motifs are selectedfrom: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, Drepresents terminal nucleosides. In certain embodiments, such terminalnucleosides are not designed to hybridize to the target nucleic acid(though one or more might hybridize by chance). In certain embodiments,the nucleobase of each D nucleoside is adenine, regardless of theidentity of the nucleobase at the corresponding position of the targetnucleic acid. In certain embodiments the nucleobase of each D nucleosideis thymine.

In certain embodiments, antisense compounds, including thoseparticularly suited for use as ssRNA comprise modified internucleosidelinkages arranged along the oligonucleotide or region thereof in adefined pattern or modified internucleoside linkage motif. In certainembodiments, oligonucleotides comprise a region having an alternatinginternucleoside linkage motif. In certain embodiments, oligonucleotidescomprise a region of uniformly modified internucleoside linkages. Incertain such embodiments, the oligonucleotide comprises a region that isuniformly linked by phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide is uniformly linked byphosphorothioate internucleoside linkages. In certain embodiments, eachinternucleoside linkage of the oligonucleotide is selected fromphosphodiester and phosphorothioate. In certain embodiments, eachinternucleoside linkage of the oligonucleotide is selected fromphosphodiester and phosphorothioate and at least one internucleosidelinkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6phosphorothioate internucleoside linkages. In certain embodiments, theoligonucleotide comprises at least 8 phosphorothioate internucleosidelinkages. In certain embodiments, the oligonucleotide comprises at least10 phosphorothioate internucleoside linkages. In certain embodiments,the oligonucleotide comprises at least one block of at least 6consecutive phosphorothioate internucleoside linkages. In certainembodiments, the oligonucleotide comprises at least one block of atleast 8 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least 10 consecutive phosphorothioate internucleoside linkages. Incertain embodiments, the oligonucleotide comprises at least one block ofat least one 12 consecutive phosphorothioate internucleoside linkages.In certain such embodiments, at least one such block is located at the3′ end of the oligonucleotide. In certain such embodiments, at least onesuch block is located within 3 nucleosides of the 3′ end of theoligonucleotide.

Oligonucleotides having any of the various sugar motifs describedherein, may have any linkage motif. For example, the oligonucleotides,including but not limited to those described above, may have a linkagemotif selected from non-limiting the table below:

5′ most linkage Central region 3′-region PS Alternating PO/PS 6 PS PSAlternating PO/PS 7 PS PS Alternating PO/PS 8 PS

ii. siRNA Compounds

In certain embodiments, antisense compounds are double-stranded RNAicompounds (siRNA). In such embodiments, one or both strands may compriseany modification motif described above for ssRNA. In certainembodiments, ssRNA compounds may be unmodified RNA. In certainembodiments, siRNA compounds may comprise unmodified RNA nucleosides,but modified internucleoside linkages.

Several embodiments relate to double-stranded compositions wherein eachstrand comprises a motif defined by the location of one or more modifiedor unmodified nucleosides. In certain embodiments, compositions areprovided comprising a first and a second oligomeric compound that arefully or at least partially hybridized to form a duplex region andfurther comprising a region that is complementary to and hybridizes to anucleic acid target. It is suitable that such a composition comprise afirst oligomeric compound that is an antisense strand having full orpartial complementarity to a nucleic acid target and a second oligomericcompound that is a sense strand having one or more regions ofcomplementarity to and forming at least one duplex region with the firstoligomeric compound.

The compositions of several embodiments modulate gene expression byhybridizing to a nucleic acid target resulting in loss of its normalfunction. In certain embodiment, the degradation of the targeted nucleicacid is facilitated by an activated RISC complex that is formed withcompositions of the invention.

Several embodiments are directed to double-stranded compositions whereinone of the strands is useful in, for example, influencing thepreferential loading of the opposite strand into the RISC (or cleavage)complex. The compositions are useful for targeting selected nucleic acidmolecules and modulating the expression of one or more genes. In someembodiments, the compositions of the present invention hybridize to aportion of a target RNA resulting in loss of normal function of thetarget RNA.

Certain embodiments are drawn to double-stranded compositions whereinboth the strands comprises a hemimer motif, a fully modified motif, apositionally modified motif or an alternating motif. Each strand of thecompositions of the present invention can be modified to fulfil aparticular role in for example the siRNA pathway. Using a differentmotif in each strand or the same motif with different chemicalmodifications in each strand permits targeting the antisense strand forthe RISC complex while inhibiting the incorporation of the sense strand.Within this model, each strand can be independently modified such thatit is enhanced for its particular role. The antisense strand can bemodified at the 5′-end to enhance its role in one region of the RISCwhile the 3′-end can be modified differentially to enhance its role in adifferent region of the RISC.

The double-stranded oligonucleotide molecules can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The double-stranded oligonucleotide molecules can beassembled from two separate oligonucleotides, where one strand is thesense strand and the other is the antisense strand, wherein theantisense and sense strands are self-complementary (i.e. each strandcomprises nucleotide sequence that is complementary to nucleotidesequence in the other strand; such as where the antisense strand andsense strand form a duplex or double-stranded structure, for examplewherein the double-stranded region is about to about 30, e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs;the antisense strand comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense strand comprises nucleotide sequence correspondingto the target nucleic acid sequence or a portion thereof (e.g., about 15to about 25 or more nucleotides of the double-stranded oligonucleotidemolecule are complementary to the target nucleic acid or a portionthereof). Alternatively, the double-stranded oligonucleotide isassembled from a single oligonucleotide, where the self-complementarysense and antisense regions of the siRNA are linked by means of anucleic acid based or non-nucleic acid-based linker(s).

The double-stranded oligonucleotide can be a polynucleotide with aduplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The double-stranded oligonucleotide can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siRNA molecule capable of mediating RNAi.

In certain embodiments, the double-stranded oligonucleotide comprisesseparate sense and antisense sequences or regions, wherein the sense andantisense regions are covalently linked by nucleotide or non-nucleotidelinkers molecules as is known in the art, or are alternatelynon-covalently linked by ionic interactions, hydrogen bonding, van derwaals interactions, hydrophobic interactions, and/or stackinginteractions. In certain embodiments, the double-strandedoligonucleotide comprises nucleotide sequence that is complementary tonucleotide sequence of a target gene. In another embodiment, thedouble-stranded oligonucleotide interacts with nucleotide sequence of atarget gene in a manner that causes inhibition of expression of thetarget gene.

As used herein, double-stranded oligonucleotides need not be limited tothose molecules containing only RNA, but further encompasses chemicallymodified nucleotides and non-nucleotides. In certain embodiments, theshort interfering nucleic acid molecules lack 2′-hydroxy (2′-OH)containing nucleotides. In certain embodiments short interfering nucleicacids optionally do not include any ribonucleotides (e.g., nucleotideshaving a 2′-OH group). Such double-stranded oligonucleotides that do notrequire the presence of ribonucleotides within the molecule to supportRNAi can however have an attached linker or linkers or other attached orassociated groups, moieties, or chains containing one or morenucleotides with 2′-OH groups. Optionally, double-strandedoligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30,40, or 50% of the nucleotide positions. As used herein, the term siRNAis meant to be equivalent to other terms used to describe nucleic acidmolecules that are capable of mediating sequence specific RNAi, forexample short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), short hairpin RNA (shRNA), short interferingoligonucleotide, short interfering nucleic acid, short interferingmodified oligonucleotide, chemically modified siRNA,post-transcriptional gene silencing RNA (ptgsRNA), and others. Inaddition, as used herein, the term RNAi is meant to be equivalent toother terms used to describe sequence specific RNA interference, such aspost transcriptional gene silencing, translational inhibition, orepigenetics. For example, double-stranded oligonucleotides can be usedto epigenetically silence genes at both the post-transcriptional leveland the pre-transcriptional level. In a non-limiting example, epigeneticregulation of gene expression by siRNA molecules of the invention canresult from siRNA mediated modification of chromatin structure ormethylation pattern to alter gene expression (see, for example, Verdelet al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science,303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al.,2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218;and Hall et al., 2002, Science, 297, 2232-2237).

It is contemplated that compounds and compositions of severalembodiments provided herein can target by a dsRNA-mediated genesilencing or RNAi mechanism, including, e.g., “hairpin” or stem-loopdouble-stranded RNA effector molecules in which a single RNA strand withself-complementary sequences is capable of assuming a double-strandedconformation, or duplex dsRNA effector molecules comprising two separatestrands of RNA. In various embodiments, the dsRNA consists entirely ofribonucleotides or consists of a mixture of ribonucleotides anddeoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, byWO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filedApr. 21, 1999. The dsRNA or dsRNA effector molecule may be a singlemolecule with a region of self-complementarity such that nucleotides inone segment of the molecule base pair with nucleotides in anothersegment of the molecule. In various embodiments, a dsRNA that consistsof a single molecule consists entirely of ribonucleotides or includes aregion of ribonucleotides that is complementary to a region ofdeoxyribonucleotides. Alternatively, the dsRNA may include two differentstrands that have a region of complementarity to each other.

In various embodiments, both strands consist entirely ofribonucleotides, one strand consists entirely of ribonucleotides and onestrand consists entirely of deoxyribonucleotides, or one or both strandscontain a mixture of ribonucleotides and deoxyribonucleotides. Incertain embodiments, the regions of complementarity are at least 70, 80,90, 95, 98, or 100% complementary to each other and to a target nucleicacid sequence. In certain embodiments, the region of the dsRNA that ispresent in a double-stranded conformation includes at least 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or5000 nucleotides or includes all of the nucleotides in a cDNA or othertarget nucleic acid sequence being represented in the dsRNA. In someembodiments, the dsRNA does not contain any single stranded regions,such as single stranded ends, or the dsRNA is a hairpin. In otherembodiments, the dsRNA has one or more single stranded regions oroverhangs. In certain embodiments, RNA/DNA hybrids include a DNA strandor region that is an antisense strand or region (e.g, has at least 70,80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and anRNA strand or region that is a sense strand or region (e.g, has at least70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and viceversa.

In various embodiments, the RNA/DNA hybrid is made in vitro usingenzymatic or chemical synthetic methods such as those described hereinor those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No.60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strandsynthesized in vitro is complexed with an RNA strand made in vivo or invitro before, after, or concurrent with the transformation of the DNAstrand into the cell. In yet other embodiments, the dsRNA is a singlecircular nucleic acid containing a sense and an antisense region, or thedsRNA includes a circular nucleic acid and either a second circularnucleic acid or a linear nucleic acid (see, for example, WO 00/63364,filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.)Exemplary circular nucleic acids include lariat structures in which thefree 5′ phosphoryl group of a nucleotide becomes linked to the 2′hydroxyl group of another nucleotide in a loop back fashion.

In other embodiments, the dsRNA includes one or more modifiednucleotides in which the 2′ position in the sugar contains a halogen(such as fluorine group) or contains an alkoxy group (such as a methoxygroup) which increases the half-life of the dsRNA in vitro or in vivocompared to the corresponding dsRNA in which the corresponding 2′position contains a hydrogen or an hydroxyl group. In yet otherembodiments, the dsRNA includes one or more linkages between adjacentnucleotides other than a naturally-occurring phosphodiester linkage.Examples of such linkages include phosphoramide, phosphorothioate, andphosphorodithioate linkages. The dsRNAs may also be chemically modifiednucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In otherembodiments, the dsRNA contains one or two capped strands, as disclosed,for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No.60/130,377, filed Apr. 21, 1999.

In other embodiments, the dsRNA can be any of the at least partiallydsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNAmolecules described in U.S. Provisional Application 60/399,998; and U.S.Provisional Application 60/419,532, and PCT/US2003/033466, the teachingof which is hereby incorporated by reference. Any of the dsRNAs may beexpressed in vitro or in vivo using the methods described herein orstandard methods, such as those described in WO 00/63364.

Occupancy

In certain embodiments, antisense compounds are not expected to resultin cleavage or the target nucleic acid via RNase H or to result incleavage or sequestration through the RISC pathway. In certain suchembodiments, antisense activity may result from occupancy, wherein thepresence of the hybridized antisense compound disrupts the activity ofthe target nucleic acid. In certain such embodiments, the antisensecompound may be uniformly modified or may comprise a mix ofmodifications and/or modified and unmodified nucleosides.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode A1AT include, without limitation, thefollowing: GENBANK Accession No. NM_000295.4 (incorporated herein as SEQID NO: 1), the complement of GENBANK Accession No. NT_026437.12truncated from nucleosides 75840001 to 75860000 (incorporated herein asSEQ ID NO: 2), a variant sequence at the site of the PiZ mutation(incorporated herein as SEQ ID NO: 3), GENBANK Accession No.NM_001002235.2 (incorporated herein as SEQ ID NO: 4), GENBANK AccessionNo. NM_001002236.2 (incorporated herein as SEQ ID NO: 5), GENBANKAccession No. NM_001127700.1 (incorporated herein at SEQ ID NO: 6),GENBANK Accession No. NM 001127701.1 (incorporated herein as SEQ ID NO:7), GENBANK Accession No. NM_001127702.1 (incorporated herein as SEQ IDNO: 8), GENBANK Accession No. NM_001127703.1 (incorporated herein as SEQID NO: 9), GENBANK Accession No. NM_001127704.1 (incorporated herein asSEQ ID NO: 10), GENBANK Accession No. NM_001127705.1 (incorporatedherein as SEQ ID NO: 11), GENBANK Accession No. NM_001127706.1(incorporated herein as SEQ ID NO: 12), GENBANK Accession No.NM_001127707.1 (incorporated herein as SEQ ID NO: 13), and GENBANKAccession No. NW_001121215.1 truncated from nucleotides 7483001 to U.S.Pat. No. 7,503,000 (incorporated herein as SEQ ID NO: 14).

It is understood that the sequence set forth in each SEQ ID NO in theExamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No)indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region may encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor A1AT can be obtained by accession number from sequence databasessuch as NCBI and such information is incorporated herein by reference.In certain embodiments, a target region may encompass the sequence froma 5′ target site of one target segment within the target region to a 3′target site of another target segment within the same target region.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple targetsegments within a target region may be overlapping. Alternatively, theymay be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides on the target nucleicacid, or is a range defined by any two of the preceeding values. Incertain embodiments, target segments within a target region areseparated by no more than, or no more than about, 5 nucleotides on thetarget nucleic acid. In certain embodiments, target segments arecontiguous. Contemplated are target regions defined by a range having astarting nucleic acid that is any of the 5′ target sites or 3′ targetsites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment may specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments may include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm may be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that mayhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In certain embodiments, reductions inA1AT mRNA levels are indicative of inhibition of A1AT expression.Reductions in levels of an A1AT protein are also indicative ofinhibition of target mRNA expression. Further, phenotypic changes areindicative of inhibition of A1AT expression. For example, reduced orprevented A1AT protein aggregates can be indicative of inhibition ofA1AT expression. In another example, prevented liver dysfunction can beindicative of inhibition of A1AT expression. In another example,restored liver function can be indicative of inhibition of A1 ATexpression. In another example, prevented or reduced hepatic toxicitycan be indicative of A1AT expression. In another example, preventedpulmonary dysfunction can be indicative of inhibition of A1ATexpression. In another example, restored pulmonary function can beindicative of inhibition of A1AT expression. In another example,prevented or reduced pulmonary toxicity can be indicative of A1ATexpression.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and an A1AT nucleic acid. The most common mechanism ofhybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteenor reversed Hoogsteen hydrogen bonding) between complementarynucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art. In certainembodiments, the antisense compounds provided herein are specificallyhybridizable with an A1AT nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as an A1AT nucleicacid).

Non-complementary nucleobases between an antisense compound and an A1ATnucleic acid may be tolerated provided that the antisense compoundremains able to specifically hybridize to a target nucleic acid.Moreover, an antisense compound may hybridize over one or more segmentsof an A1AT nucleic acid such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%complementary to an A1AT nucleic acid, a target region, target segment,or specified portion thereof. Percent complementarity of an antisensecompound with a target nucleic acid can be determined using routinemethods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403410; Zhang and Madden,Genome Res., 1997, 7, 649656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e., 100%complementary) to a target nucleic acid, or specified portion thereof.For example, an antisense compound may be fully complementary to an A1ATnucleic acid, or a target region, or a target segment or target sequencethereof. As used herein, “fully complementary” means each nucleobase ofan antisense compound is capable of precise base pairing with thecorresponding nucleobases of a target nucleic acid. For example, a 20nucleobase antisense compound is fully complementary to a targetsequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and/or thesecond nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound may or may not be fully complementary tothe target sequence, depending on whether the remaining 10 nucleobasesof the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases may be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they may be contiguous (i.e., linked) or non-contiguous. In oneembodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 12,13, 14, 15, 16, 17, 18, 19, or nucleobases in length comprise no morethan 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas an A1AT nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas an A1AT nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 12 nucleobase portion of a target segment.In certain embodiments, the antisense compounds are complementary to atleast a 15 nucleobase portion of a target segment. In certainembodiments, the antisense compounds are complementary to at least an 18nucleobase portion of a target segment. Also contemplated are antisensecompounds that are complementary to at least a 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment,or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or compoundrepresented by a specific Isis number, or portion thereof. As usedherein, an antisense compound is identical to the sequence disclosedherein if it has the same nucleobase pairing ability. For example, a RNAwhich contains uracil in place of thymidine in a disclosed DNA sequencewould be considered identical to the DNA sequence since both uracil andthymidine pair with adenine. Shortened and lengthened versions of theantisense compounds described herein as well as compounds havingnon-identical bases relative to the antisense compounds provided hereinalso are contemplated. The non-identical bases may be adjacent to eachother or dispersed throughout the antisense compound. Percent identityof an antisense compound is calculated according to the number of basesthat have identical base pairing relative to the sequence to which it isbeing compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to one or more of the antisense compounds or SEQ ID NOs, or aportion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is comparedto an equal length portion of the target nucleic acid. In certainembodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleobase portion is compared to an equal lengthportion of the target nucleic acid. In certain embodiments, a portion ofthe antisense oligonucleotide is compared to an equal length portion ofthe target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portionis compared to an equal length portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occuring internucleoside linkage of RNA and DNA is a 3′ to5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to an A1AT nucleicacid comprise one or more modified internucleoside linkages. In certainembodiments, the modified internucleoside linkages are phosphorothioatelinkages. In certain embodiments, each internucleoside linkage of anantisense compound is a phosphorothioate internucleoside linkage.

Modified Sugar Moieties

Antisense compounds can optionally contain one or more nucleosideswherein the sugar group has been modified. Such sugar modifiednucleosides may impart enhanced nuclease stability, increased bindingaffinity, or some other beneficial biological property to the antisensecompounds. In certain embodiments, nucleosides comprise chemicallymodified ribofuranose ring moieties. Examples of chemically modifiedribofuranose rings include without limitation, addition of substitutentgroups (including 5′ and 2′ substituent groups, bridging of non-geminalring atoms to form bicyclic nucleic acids (BNA), replacement of theribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂ areeach independently H, C₁-C₁₂ alkyl or a protecting group) andcombinations thereof. Examples of chemically modified sugars include2′-F-5′-methyl substituted nucleoside (see PCT International ApplicationWO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atomwith S with further substitution at the 2′-position (see published U.S.Patent Application US2005-0130923, published on Jun. 16, 2005) oralternatively 5′-substitution of a BNA (see PCT InternationalApplication WO 2007/134181 Published on Nov. 22, 2007 wherein LNA issubstituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′-OCH₂CH₂F and 2′-O(CH₂)₂—OCH₃ substituentgroups. The substituent at the 2′ position can also be selected fromallyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), andO—CH₂—C(═O)—N(R₁)—(CH₂)₂—N(R_(m))(R₁₁), where each R₁, R_(m) and R_(n)is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleosidesinclude without limitation nucleosides comprising a bridge between the4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisensecompounds provided herein include one or more bicyclic nucleosidescomprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclicnucleosides, include but are not limited to one of the formulae:4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA);4′-CH(CH₃)—O-2′ (also referred to as constrained ethyl or cEt) and4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845,issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof seepublished International Application WO/2009/006478, published Jan. 8,2009); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof see publishedInternational Application WO/2008/150729, published Dec. 11, 2008);4′-CH₂—O—N(CH₃)-2′ (see published U.S. Patent ApplicationUS2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′, wherein R isH, C₁-C₁₂ alkyl, or a protecting group (see U.S. Pat. No. 7,427,672,issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (andanalogs thereof see published International Application WO 2008/154401,published on Dec. 8, 2008).

Further reports related to bicyclic nucleosides can also be found inpublished literature (see for example: Singh et al., Chem. Commun.,1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630;Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638;Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh etal., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am.Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. OpinionInvest. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8,1-7; and Oram et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S.Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499;7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. PatentPublication No. US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos.60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787;and 61/099,844; Published PCT International applications WO 1994/014226;WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO2008/154401; and WO 2009/006478. Each of the foregoing bicyclicnucleosides can be prepared having one or more stereochemical sugarconfigurations including for example α-L-ribofuranose andβ-D-ribofuranose (see PCT international application PCT/DK98/00393,published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ position of the pentofuranosyl sugar moietywherein such bridges independently comprises 1 or from 2 to 4 linkedgroups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═O)—, —C(═NR_(a))—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is—[C(R_(a))(R_(b))]_(n)—, —C(R_(a))(R_(b))_(n)—O—, —C(R_(a)R_(b))—N(R)—O—or —C(R_(a)R_(b))—O—N(R)—. In certain embodiments, the bridge is4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′,4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′- wherein each R is, independently,H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 27, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methyleneoxy(4′-CH₂—O-2′) BNA, (C) ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, and (F)methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene-thio(4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methylcarbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic(4′-(CH₂)₃-2′) BNA and (K) vinyl BNA as depicted below:

wherein Bx is the base moiety and R is independently H, a protectinggroup, C₁-C₁₂ alkyl or C₁-C₁₂ alkoxy.

In certain embodiments, bicyclic nucleosides are provided having FormulaI:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides are provided having FormulaII:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thio.

In one embodiment, each of the substituted groups is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(c)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleosides are provided having FormulaIII:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides are provided having FormulaIV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides are provided having FormulaV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j),SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k),C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630). BNAs and preparation thereof are also described in WO98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA and 2′-thio-BNAs, have alsobeen prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs comprisingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., WO 99/14226).Furthermore, synthesis of 2′-amino-BNA, a novel comformationallyrestricted high-affinity oligonucleotide analog has been described inthe art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Inaddition, 2′-amino- and 2′-methylamino-BNA's have been prepared and thethermal stability of their duplexes with complementary RNA and DNAstrands has been previously reported.

In certain embodiments, bicyclic nucleosides are provided having FormulaVI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(l) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Freier etal., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al, J. Am.Chem. Soc., 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting two carbon atoms of the furanose ringconnects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

As used herein, “monocylic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In certainembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In certain embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In certain embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F,O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃ and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, wheren and m are from 1 to about 10. Other 2′-substituent groups can also beselected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving pharmacokinetic properties, or a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In certain embodiments, modifiednucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem.,1997, 272, 11944-12000). Such 2′-MOE substitution have been described ashaving improved binding affinity compared to unmodified nucleosides andto other modified nucleosides, such as 2′-O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-MOE substituent also havebeen shown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504;Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc.Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,1997, 16, 917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THPnucleoside” means a nucleoside having a six-membered tetrahydropyran“sugar” substituted in for the pentofuranosyl residue in normalnucleosides (a sugar surrogate). Modified THP nucleosides include, butare not limited to, what is referred to in the art as hexitol nucleicacid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA)having a tetrahydropyran ring system as illustrated below:

In certain embodiments, sugar surrogates are selected having FormulaVII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VIE

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, an internucleoside linkinggroup linking the tetrahydropyran nucleoside analog to the antisensecompound or one of T_(a) and T_(b) is an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundand the other of T_(a) and T_(b) is H, a hydroxyl protecting group, alinked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is selectedfrom hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein Xis O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII areprovided wherein one of R and R₂ is fluoro. In certain embodiments, R isfluoro and R₂ is H; R is methoxy and R₂ is H, and R is methoxyethoxy andR₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example nucleosides comprisingmorpholino sugar moieties and their use in oligomeric compounds has beenreported (see for example: Braasch et al., Biochemistry, 2002, 41,4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and5,034,506). As used here, the term “morpholino” means a sugar surrogatehaving the following formula:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides) and replacement of theribosyl ring oxygen atom with S and further substitution at the2′-position (see published U.S. Patent Application US2005-0130923,published on Jun. 16, 2005) or alternatively 5′-substitution of abicyclic nucleic acid (see PCT International Application WO 2007/134181,published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside isfurther substituted at the 5′ position with a 5′-methyl or a 5′-vinylgroup). The synthesis and preparation of carbocyclic bicyclicnucleosides along with their oligomerization and biochemical studieshave also been described (see, e.g., Srivastava et al., J. Am. Chem.Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or moremodified cyclohexenyl nucleosides, which is a nucleoside having asix-membered cyclohexenyl in place of the pentofuranosyl residue innaturally occurring nucleosides. Modified cyclohexenyl nucleosidesinclude, but are not limited to those described in the art (see forexample commonly owned, published PCT Application WO 2010/036696,published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008,130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48,3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30),9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005,24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005,33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F:Structural Biology and Crystallization Communications, 2005, F61(6),585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al.,Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem.,2003, 68. 4499-4505; Verbeure et al., Nucleic Acids Research, 2001,29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wanget al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7),785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCTapplication, WO 06/047842; and Published PCT Application WO 01/049687;the text of each is incorporated by reference herein, in theirentirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenylnucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the cyclohexenyl nucleoside analog to an antisense compound orone of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to an antisense compound and the otherof T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugarsubstituent group.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to anucleoside comprising a sugar comprising an —OCH₃ group at the 2′position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′-OCH₂CH₂OCH₃” or“2′-O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a —OCH₂CH₂OCH₃ group at the 2′ position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see for example review article:Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems canundergo various additional substitutions to enhance activity.

Methods for the preparations of modified sugars are well known to thoseskilled in the art. Some representative U.S. patents that teach thepreparation of such modified sugars include without limitation, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,670,633; 5,700,920; 5,792,847 and 6,600,032 and InternationalApplication PCT/US2005/019219, filed Jun. 2, 2005 and published as WO2005/121371 on Dec. 22, 2005, and each of which is herein incorporatedby reference in its entirety.

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleosides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleosides are arranged in a gapmer motif. In certainembodiments, the modified sugar moiety is a bicyclic nucleoside having a(4′-CH(CH₃)—O-2) bridging group. In certain embodiments, the(4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wingsof a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases. Both natural and modifiednucleobases are capable of participating in hydrogen bonding. Suchnucleobase modifications can impart nuclease stability, binding affinityor some other beneficial biological property to antisense compounds.Modified nucleobases include synthetic and natural nucleobases such as,for example, 5-methylcytosine (5-me-C). Certain nucleobasesubstitutions, including 5-methylcytosine substitutions, areparticularly useful for increasing the binding affinity of an antisensecompound for a target nucleic acid. For example, 5-methylcytosinesubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties can also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of antisense compounds include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds comprise one or moremodified nucleobases. In certain embodiments, shortened or gap-widenedantisense oligonucleotides comprise one or more modified nucleobases. Incertain embodiments, the modified nucleobase is 5-methylcytosine. Incertain embodiments, each cytosine is a 5-methylcytosine.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides may be admixed with pharmaceuticallyacceptable active or inert substances for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

An antisense compound targeted to an A1AT nucleic acid can be utilizedin pharmaceutical compositions by combining the antisense compound witha suitable pharmaceutically acceptable diluent or carrier. Apharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredparenterally or by inhalation. Accordingly, in one embodiment, employedin the methods described herein is a pharmaceutical compositioncomprising an antisense compound targeted to an A1AT nucleic acid and apharmaceutically acceptable diluent. In certain embodiments, thepharmaceutically acceptable diluent is PBS. In certain embodiments, theantisense compound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to pharmaceutically acceptablesalts of antisense compounds, prodrugs, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acid from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

In certain embodiments, antisense compounds, including, but not limitedto those particularly suited for use as ssRNA, are modified byattachment of one or more conjugate groups. In general, conjugate groupsmodify one or more properties of the attached oligonucleotide, includingbut not limited to pharmacodynamics, pharmacokinetics, stability,binding, absorption, cellular distribution, cellular uptake, charge andclearance. Conjugate groups are routinely used in the chemical arts andare linked directly or via an optional conjugate linking moiety orconjugate linking group to a parent compound such as an oligonucleotide.Conjugate groups includes without limitation, intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, thioethers,polyethers, cholesterols, thiocholesterols, cholic acid moieties,folate, lipids, phospholipids, biotin, phenazine, phenanthridine,anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarinsand dyes. Certain conjugate groups have been described previously, forexample: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-aminonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

For additional conjugates including those useful for ssRNA and theirplacement within antisense compounds, see e.g., U.S. Application No.61/583,963.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof A1AT nucleic acids can be tested in vitro in a variety of cell types.Cell types used for such analyses are available from commercial vendors(e.g. American Type Culture Collection, Manassas, Va.; Zen-Bio, Inc.,Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.)and are cultured according to the vendor's instructions usingcommercially available reagents (e.g. Invitrogen Life Technologies,Carlsbad, Calif.). Illustrative cell types include, but are not limitedto, HepG2 cells, Hep3B cells, and transgenic mouse primary hepatocytes.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a LIPOFECTAMINEconcentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation.

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein. In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art. Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM when transfected withLIPOFECTAMINE. Antisense oligonucleotides are used at higherconcentrations ranging from 625 to 20,000 nM when transfected usingelectroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+mRNA.Methods of RNA isolation are well known in the art. RNA is preparedusing methods well known in the art, for example, using the TRIZOLReagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of an A1AT nucleic acid can beassayed in a variety of ways known in the art. For example, targetnucleic acid levels can be quantitated by, e.g., Northern blot analysis,competitive polymerase chain reaction (PCR), or quantitative real-timePCR. RNA analysis can be performed on total cellular RNA orpoly(A)+mRNA. Methods of RNA isolation are well known in the art.Northern blot analysis is also routine in the art. Quantitativereal-time PCR can be conveniently accomplished using the commerciallyavailable ABI PRISM 7600, 7700, or 7900 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence DetectionSystem (PE-Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions. Methods of quantitative real-time PCR arewell known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carriedout by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expressionis quantified by real time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA is quantified usingRIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000instrument (PE Applied Biosystems) is used to measure RIBOGREENfluorescence.

Probes and primers are designed to hybridize to an A1AT nucleic acid.Methods for designing real-time PCR probes and primers are well known inthe art, and may include the use of software such as PRIMER EXPRESSSoftware (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of A1AT nucleic acids can be assessed by measuringA1AT protein levels. Protein levels of A1AT can be evaluated orquantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting),enzyme-linked immunosorbent assay (ELISA), quantitative protein assays,protein activity assays (for example, caspase activity assays),immunohistochemistry, immunocytochemistry or fluorescence-activated cellsorting (FACS). Antibodies directed to a target can be identified andobtained from a variety of sources, such as the MSRS catalog ofantibodies (Aerie Corporation, Birmingham, Mich.), or can be preparedvia conventional monoclonal or polyclonal antibody generation methodswell known in the art. Antibodies useful for the detection of mouse,rat, monkey, and human A1AT are commercially available.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression of A1AT andproduce phenotypic changes, such as, reduced or prevented A1AT proteinaggregation, prevented liver and/or pulmonary dysfunction, restoredliver and/or pulmonary function, prevented or reduced hepatic and/orpulmonary toxicity. Such parameters may be indicative of A1ATexpression. In certain embodiments, A1ATD associated liver dysfunctionor hepatic toxicity is determined by measuring A1AT aggregates in livertissue, measuring transaminases (including ALT and AST), measuringbilirubin, and serum albumin. In certain embodiments, A1ATD associatedpulmonary dysfunction or pulmonary toxicity is determined by measuringATAT aggregates in pulmonary tissue or by spirometry to measure FEV₁ andFVC. Testing may be performed in normal animals, or in experimentaldisease models. For administration to animals, antisenseoligonucleotides are formulated in a pharmaceutically acceptablediluent, such as phosphate-buffered saline. Administration includesparenteral routes of administration, such as intraperitoneal,intravenous, and subcutaneous. Administration also includes pulmonaryroutes of administration, such as nebulization, inhalation, andinsufflation. Calculation of antisense oligonucleotide dosage and dosingfrequency depends upon factors such as route of administration andanimal body weight. Following a period of treatment with antisenseoligonucleotides, RNA is isolated from liver tissue and/or pulmonarytissue and changes in A1AT nucleic acid expression are measured.

Certain Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions described herein. In certain embodiments, the individualhas a liver disease, such as A1ATD associated liver disease. In certainembodiments, the individual is at risk for developing A1ATD associatedliver disease. This includes individuals with a genetic predispositionto developing A1ATD. In certain embodiments, the individual has beenidentified as in need of therapy. Examples of such individuals include,but are not limited to those having a mutation in the genetic code forA1 AT. In certain embodiments, provided herein are methods forprophylactically reducing A1AT expression in an individual. Certainembodiments include treating an individual in need thereof byadministering to an individual a therapeutically effective amount of anantisense compound targeted to an A1AT nucleic acid.

In one embodiment, administration of a therapeutically effective amountof an antisense compound targeted to an A1AT nucleic acid is accompaniedby monitoring of A1AT levels in the individual, to determine anindividual's response to administration of the antisense compound. Anindividual's response to administration of the antisense compound isused by a physician to determine the amount and duration of therapeuticintervention.

In certain embodiments, administration of an antisense compound targetedto an A1AT nucleic acid results in reduction of A1AT expression by atleast 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95or 99%, or a range defined by any two of these values. In certainembodiments, administration of an antisense compound targeted to an A1ATnucleic acid results in a change in a measure of A1AT aggregatesretained in the liver, liver function, and hepatic toxicity. In certainembodiments, administration of an A1AT antisense compound decreases themeasure by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95 or 99%, or a range defined by any two of these values. Insome embodiments, administration of an A1AT antisense compound increasesthe measure by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or 99%, or a range defined by any two of thesevalues.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to A1AT are used for the preparation of amedicament for treating a patient suffering or susceptible to a liverdisease, such as, A1ATD associated liver disease.

Certain Combination Therapies

In certain embodiments, one or more pharmaceutical compositionsdescribed herein are co-administered with one or more otherpharmaceutical agents. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat the same disease, disorder,or condition as the one or more pharmaceutical compositions describedherein. In certain embodiments, such one or more other pharmaceuticalagents are designed to treat a different disease, disorder, or conditionas the one or more pharmaceutical compositions described herein. Incertain embodiments, such one or more other pharmaceutical agents aredesigned to treat an undesired side effect of one or more pharmaceuticalcompositions described herein. In certain embodiments, one or morepharmaceutical compositions described herein are co-administered withanother pharmaceutical agent to treat an undesired effect of that otherpharmaceutical agent. In certain embodiments, one or more pharmaceuticalcompositions described herein are co-administered with anotherpharmaceutical agent to produce a combinational effect. In certainembodiments, one or more pharmaceutical compositions described hereinare co-administered with another pharmaceutical agent to produce asynergistic effect.

In certain embodiments, one or more pharmaceutical compositionsdescribed herein and one or more other pharmaceutical agents areadministered at the same time. In certain embodiments, one or morepharmaceutical compositions described herein and one or more otherpharmaceutical agents are administered at different times. In certainembodiments, one or more pharmaceutical compositions described hereinand one or more other pharmaceutical agents are prepared together in asingle formulation. In certain embodiments, one or more pharmaceuticalcompositions described herein and one or more other pharmaceuticalagents are prepared separately.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition described hereininclude carbamazepine, A1AT replacement therapy, antiviral therapy,lipid lowering therapy, steroids and COPD therapies. In certainembodiments, antiviral therapy includes interferon alpha-2b, interferonalpha-2a, and interferon alphacon-1 (pegylated and unpegylated);ribavirin; penciclovir (Denavir); foscamet (Foscavir); Ascoxal;Acyclovir; Valacyclovir; Famciclovir; a viral RNA replication inhibitor;a second antisense oligomer; a viral therapeutic vaccine; a viralprophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovirdiisoproxil fumarate (TDF); telbivudine (LdT); adefovir; or ananti-virus antibody therapy (monoclonal or polyclonal). In certainembodiments, lipid lowering therapy includes, but is not limited to,bile salt sequestering resins (e.g., cholestyramine, colestipol, andcolesevelam hydrochloride), cholesterol biosynthesis inhibitors,especially HMG CoA reductase inhibitors (such as atorvastatin,pravastatin, simvastatin, lovastatin, fluvastatin, cerivastatin,rosuvastatin, and pitivastatin (itavastatin/risivastatin)), nicotinicacid, fibric acid derivatives (e.g., clofibrate, gemfibrozil,fenofibrate, bezafibrate, and ciprofibrate), probucol, neomycin,dextrothyroxine, plant-stanol esters, cholesterol absorption inhibitors(e.g., ezetimibe and pamaqueside), CETP inhibitors (e.g. torcetrapib,and JTT-705) MTP inhibitors (e.g., implitapide), squalene synthetaseinhibitors, bile acid sequestrants such as cholestyramine, inhibitors ofbile acid transporters (apical sodium-dependent bile acid transporters),regulators of hepatic CYP7a, ACAT inhibitors (e.g. Avasimibe), estrogenreplacement therapeutics (e.g., tamoxigen), synthetic HDL (e.g.ETC-216), anti-inflammatories (e.g., glucocorticoids) and antisensecompounds targeting cardiovascular targets (e.g., Apo B targetingcompounds and ApoC-III targeting compounds). In certain embodiments,COPD therapies include, for example, anti-inflammation drugs;bronchodilators, such as ipratropium (Atrovent), tiotropium (Spiriva),salmeterol (Serevent), formoterol (Foradil), or albuterol; or oxygentherapy. Anti-inflammatory drugs can include steroids, NSAIDS(non-steroidal anti-inflammatory drugs), COX inhibitors, montelukast(Singulair), roflimulast, antihistamines and the like. In certainembodiments, the second agent can be an asthma drug such as ananti-inflammatory drug, a bronchodilator (e.g., beta-2 agonists (LABA2),theophylline, ipratropium), a leukotriene modifier, Cromolyn,nedocromil, a decongestant and immunotherapy. In certain embodiments,such co-administration is for the treatment of liver disease. In certainembodiments, such co-administration is for the treatment of pulmonarydisease.

In certain embodiments, pharmaceutical agents that may beco-administered with an A1AT specific inhibitor as described hereininclude, but are not limited to, an additional A1AT inhibitor. Incertain embodiments, the co-adminstered pharmaceutical agent isadministered prior to administration of a pharmaceutical compositiondescribed herein. In certain embodiments, the co-administeredpharmaceutical agent is administered following administration of apharmaceutical composition described herein. In certain embodiments theco-administered pharmaceutical agent is administered at the same time asa pharmaceutical composition described herein. In certain embodimentsthe dose of a co-administered pharmaceutical agent is the same as thedose that would be administered if the co-administered pharmaceuticalagent was administered alone. In certain embodiments the dose of aco-administered pharmaceutical agent is lower than the dose that wouldbe administered if the co-administered pharmaceutical agent wasadministered alone. In certain embodiments the dose of a co-administeredpharmaceutical agent is greater than the dose that would be administeredif the co-administered pharmaceutical agent was administered alone.

In certain embodiments, the co-administration of a second compoundenhances the effect of a first compound, such that co-administration ofthe compounds results in an effect that is greater than the effect ofadministering the first compound alone. In other embodiments, theco-administration results in an effect that is additive of the effectsof the compounds when administered alone. In certain embodiments, theco-administration results an effect that is supra-additive of the effectof the compounds when administered alone. In certain embodiments, thefirst compound is an antisense compound. In certain embodiments, thesecond compound is an antisense compound.

Certain Compounds

Approximately 700 modified antisense oligonucleotides were tested fortheir effect on human A1AT mRNA in vitro in several cell types. Of theapproximately 700 modified antisense oligonucleotides, twenty-threecompounds were selected for further in vitro studies based on in vitroactivity to test their potency in dose response studies in PiZtransgenic mice primary hepatocytes, HepG2 cells, Hep3B cells. Of thetwenty-three compound, fifteen compounds were tested in CD1 mice andSprague-Dawley rats for tolerability, and in PiZ transgenic mice forefficacy and tolerability. A final selection of seven compounds was madefor further study in cynomolgous monkeys based on systemic tolerabilityand activity in the rodent studies. These seven compounds were selectedbecause they are highly tolerable and very active in transgenic PiZmice. The compounds are complementary to the regions 1421-1440,1561-1580, 1564-1583, 1565-1584, 1571-1590, 1575-1594, and 1577-1596 ofSEQ ID NO: 1. In certain embodiments, the compounds targeting the listedregions comprise a modified oligonucleotide having some nucleobaseportion of the sequence recited in SEQ ID NOs: 23, 26, 29, 30, 34, 38,and 40. In certain embodiments, the compounds targeting the listedregions or having a nucleobase portion of a sequence recited in thelisted SEQ ID NOs can be various lengths and may have one of variousmotifs. In certain embodiments, a compound targeting a region or havinga nucleobase portion of a sequence recited in the listed SEQ ID NOs hasthe specific length and motif as indicated by the ISIS NOs: 487660,487662, 496386, 496392, 496393, 496404, and 496407. Compounds describedabove as being highly tolerable and active in transgenic PiZ mice weretested in cynomologous monkeys to assess tolerability in a primate.

In certain embodiments, the compounds as described herein areefficacious by virtue of having at least one of an in vitro IC₅₀ of lessthan 10 μM, less than 9 μM, less than 8 μM, less than 7 μM, less than 6μM, less than 5 μM, less than 4 μM, less than 3 μM, less than 2 μM, lessthan 1 μM when delivered to a human cell line as described herein. Incertain embodiments, the compounds as described herein are highlytolerable as demonstrated by having at least one of an increase an ALTor AST value of no more than 4 fold, 3 fold, or 2 fold over salinetreated animals or an increase in liver, spleen or kidney weight of nomore than 30%, 20%, 15%, 12%, 10%, 5% or 2%.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions, and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1: Antisense Inhibition of Human Alpha-1 Antitrypsin in HepG2Cells

Antisense oligonucleotides were designed targeting a human alpha-1antitrypsin (A1AT) nucleic acid and were tested for their effects onA1AT mRNA in vitro. Cultured human HepG2 cells at a density of 20,000cells per well were transfected using electroporation with 4,500 nMantisense oligonucleotide. After a treatment period of approximately 24hours, RNA was isolated from the cells and A1AT mRNA levels weremeasured by quantitative real-time PCR using human primer probe setRTS3320 (forward sequence GGAGATGCTGCCCAGAAGAC, designated herein as SEQID NO: 45; reverse sequence GCTGGCGGTATAGGCTGAAG, designated herein asSEQ ID NO: 46; probe sequence ATCAGGATCACCCAACCTTCAACAAGATCA, designatedherein as SEQ ID NO: 47). A1AT mRNA levels were adjusted according tototal RNA content, as measured by RIBOGREEN®. Results are presented aspercent inhibition of A1AT, relative to untreated control cells. Of the695 oligonucleotides tested, only those selected for further study arepresented.

The modified antisense oligonucleotides in Table 1 were designed as5-10-5 MOE gapmers. The gamers are 20 nucleosides in length, wherein thecentral gap segment comprises ten 2′-deoxynucleosides and is flanked onboth sides (in the 5′ and 3′ directions) by wings comprising fivenucleosides each. Each nucleoside in the 5′ wing segment and eachnucleoside in the 3′ wing segment has a 2′MOE sugar modification. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytosine residues throughout each gapmer are5-methylcytosines. “Human Target start site” indicates the 5′-mostnucleoside to which the gapmer is targeted in the human gene sequence.The gapmers of Table 1 are targeted to SEQ ID NO: 1 (GENBANK AccessionNo. NM_000295.4) and SEQ ID NO: 2 (the complement of GENBANK AccessionNo. NT_026437.12 truncated from nucleosides 75840001 to 75860000).

TABLE 1 Inhibition of human A1AT mRNA levels by modified antisense oligonucleotides targeted to SEQ ID NOs: 1 and 2 Start StopStart Stop Site Site Site Site on SEQ on SEQ on SEQ on SEQ ISIS % SEQID NO: 1 ID NO: 1 ID NO: 2 ID NO: 2 Motif Sequence No inhibition ID NO 459  478 10624 10643 5-10-5 TGGTGCTGTTGGACT 489009 36 20 GGTGT  464 483 10629 10648 5-10-5 GATATTGGTGCTGTT 489010 30 21 GGACT  494  51310659 10678 5-10-5 GGCTGTAGCGATGC 489013 61 22 TCACTG 1421 1440 1511815137 5-10-5 GGGTTTGTTGAACTT 496393 38 23 GACCT 1479 1498 15176 151955-10-5 CCACTTTTCCCATGA 496346 51 24 AGAGG 1493 1512 15190 15209 5-10-5TTGGGTGGGATTCA 496360 42 25 CCACTT 1561 1580 15258 15277 5-10-5CTTTAATGTCATCCA 496404 90 26 GGGAG 1562 1581 15259 15278 5-10-5TCTTTAATGTCATCC 496405 86 27 AGGGA 1563 1582 15260 15279 5-10-5TTCTTTAATGTCATC 496406 89 28 CAGGG 1564 1583 15261 15280 5-10-5CTTCTTTAATGTCAT 496407 94 29 CCAGG 1565 1584 15262 15281 5-10-5CCTTCTTTAATGTCA 496386 95 30 TCCAG 1566 1585 15263 15282 5-10-5CCCTTCTTTAATGTC 496387 97 31 ATCCA 1567 1586 15264 15283 5-10-5ACCCTTCTTTAATGT 496388 95 32 CATCC 1570 1589 15267 15286 5-10-5TCAACCCTTCTTTAA 496391 83 33 TGTCA 1571 1590 15268 15287 5-10-5CTCAACCCTTCTTTA 496392 74 34 ATGTC 1572 1591 15269 15288 5-10-5GCTCAACCCTTCTTT 487657 79 35 AATGT 1573 1592 15270 15289 5-10-5AGCTCAACCCTTCTT 487658 77 36 TAATG 1574 1593 15271 15290 5-10-5CAGCTCAACCCTTCT 487659 77 37 TTAAT 1575 1594 15272 15291 5-10-5CCAGCTCAACCCTTC 487660 87 38 TTTAA 1576 1595 15273 15292 5-10-5ACCAGCTCAACCCT 487661 83 39 TCTTTA 1577 1596 15274 15293 5-10-5GACCAGCTCAACCC 487662 79 40 TTCTTT 1578 1597 15275 15294 5-10-5GGACCAGCTCAACC 474061 84 41 CTTCTT

Example 2: Antisense Inhibition of Human Alpha-1 Antitrypsin inTransgenic Mouse Primary Hepatocytes

Transgenic mouse primary hepatocytes are from PiZ mice, originallygenerated by Sifers et al (Nucl. Acids Res. 15: 1459-1457, 1987) byintroducing a 14.4 kb DNA fragment containing the entire A1AT gene plus2 kb of 5′ and 3′ flanking genomic DNA sequences into the germ line.Primary hepatocytes were isolated from the mice and cultured for invitro screening.

Additional antisense oligonucleotides were designed targeting a humanalpha-1 antitrypsin (A1AT) nucleic acid and were tested for theireffects on A1AT mRNA in vitro. Antisense oligonucleotides from the studydescribed in Example 1 were also included in the assay and are presentedin Table 2. Cultured transgenic mouse primary hepatocytes at a densityof 10,000 cells per well were transfected using Cytofectin reagent with150 nM antisense oligonucleotide. After a treatment period ofapproximately 24 hours, RNA was isolated from the cells and A1AT mRNAlevels were measured by quantitative real-time PCR using human primerprobe set RTS3320. A1AT mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of A1AT, relative to untreated control cells. Of the 311oligonucleotides tested, only those only those selected for furtherstudy are presented.

The modified antisense oligonucleotides presented in Table 2 weredesigned as 5-10-5 MOE gapmers or 5-9-5 MOE gapmers. The 5-10-5 gapmeris 20 nucleosides in length, wherein the central gap segment comprisesof ten 2′-deoxynucleosides and is flanked on both sides (in the 5′ and3′ directions) by wings comprising five nucleosides each. The 5-9-5gapmer is 19 nucleosides in length, wherein the central gap segmentcomprises nine 2′-deoxynucleosides and is flanked on both sides (in the5′ and 3′ directions) by wings comprising five nucleosides each. Eachnucleoside in the 5′ wing segment and each nucleoside in the 3′ wingsegment has a 2′MOE sugar modification. The internucleoside linkagesthroughout the gapmer are phosphorothioate (P═S) linkages. All cytosineresidues throughout the gapmer are 5-methylcytosines. “Human Targetstart site” indicates the 5′-most nucleoside to which the gapmer istargeted in the human gene sequence. The gapmers of Table 2 are targetedto SEQ ID NO: 2 or a variant sequence, designated herein as SEQ ID NO:3.

TABLE 2 Inhibition of human A1AT mRNA levels by modified antisense oligonucleotides targeted to SEQ ID NO: 2 Start Stop StartStop Site Site Site Site on SEQ on SEQ on SEQ on SEQ ISIS % SEQ ID NO: 2ID NO: 2 ID NO: 3 ID NO: 3 No Sequence Motif inhibition ID NO 1527515294 n/a n/a 474061 GGACCAGCTCAACCCTTCTT 5-10-5 96 41 15269 15288 n/an/a 487657 GCTCAACCCTTCTTTAATGT 5-10-5 96 35 15270 15289 n/a n/a 487658AGCTCAACCCTTCTTTAATG 5-10-5 95 36 15271 15290 n/a n/a 487659CAGCTCAACCCTTCTTTAAT 5-10-5 96 37 15272 15291 n/a n/a 487660CCAGCTCAACCCTTCTTTAA 5-10-5 95 38 15273 15292 n/a n/a 487661ACCAGCTCAACCCTTCTTTA 5-10-5 95 39 15274 15293 n/a n/a 487662GACCAGCTCAACCCTTCTTT 5-10-5 95 40 10624 10643 n/a n/a 489009TGGTGCTGTTGGACTGGTGT 5-10-5 93 20 10629 10648 n/a n/a 489010GATATTGGTGCTGTTGGACT 5-10-5 90 21 10659 10678 n/a n/a 489013GGCTGTAGCGATGCTCACTG 5-10-5 94 22 15176 15195 n/a n/a 496346CCACTTTTCCCATGAAGAGG 5-10-5 95 24 15190 15209 n/a n/a 496360TTGGGTGGGATTCACCACTT 5-10-5 96 25 15262 15281 n/a n/a 496386CCTTCTTTAATGTCATCCAG 5-10-5 97 30 15263 15282 n/a n/a 496387CCCTTCTTTAATGTCATCCA 5-10-5 97 31 15264 15283 n/a n/a 496388ACCCTTCTTTAATGTCATCC 5-10-5 97 32 15267 15286 n/a n/a 496391TCAACCCTTCTTTAATGTCA 5-10-5 96 33 15268 15287 n/a n/a 496392CTCAACCCTTCTTTAATGTC 5-10-5 96 34 15118 15137 n/a n/a 496393GGGTTTGTTGAACTTGACCT 5-10-5 96 23 15258 15277 n/a n/a 496404CTTTAATGTCATCCAGGGAG 5-10-5 99 26 15259 15278 n/a n/a 496405TCTTTAATGTCATCCAGGGA 5-10-5 97 27 15260 15279 n/a n/a 496406TTCTTTAATGTCATCCAGGG 5-10-5 98 28 15261 15280 n/a n/a 496407CTTCTTTAATGTCATCCAGG 5-10-5 98 29 n/a n/a 19 37 489112GTCCCTTTCTTGTCGATGG 5-9-5 56 42

Example 3: Dose-Dependent Antisense Inhibition of Human A1AT inTransgenic Mouse Primary Hepatocytes

Transgenic mouse primary hepatocytes are from PiZ mice, originallygenerated by Sifers et al (Nucl. Acids Res. 15: 1459-1457, 1987) byintroducing a 14.4 kb DNA fragment containing the entire A1AT gene plus2 kb of 5′ and 3′ flanking genomic DNA sequences into the germ line.Primary hepatocytes were isolated from the mice and cultured for invitro screening.

Gapmers from Examples 1 and 2 exhibiting in vitro inhibition of humanA1AT were tested at various doses in transgenic mouse primaryhepatocytes. Cells were plated at a density of 10,000 cells per well andtransfected using Cytofectin reagent with 4.69 nM, 9.38 nM, 18.75 nM,37.50 nM, 75.00 nM, and 150.00 nM concentrations of antisenseoligonucleotide, as specified in Table 3. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells and A1AT mRNAlevels were measured by quantitative real-time PCR. Human A1AT primerprobe set RTS3320 was used to measure mRNA levels. A1AT mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of A1AT, relative tountreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 3. A1AT mRNA levels were reduced in adose-dependent manner in some of the antisense oligonucleotide treatedcells, ‘n.d.’ indicates that the IC₅₀ for that compound was notcalculated.

TABLE 3 Dose-dependent antisense inhibition of human A1AT in transgenicmouse primary hepatocytes ISIS 9.38 37.5 150.0 IC₅₀ No 4.69 nM nM 18.75nM nM 75.0 nM nM (nM) 474061 3 11 15 36 56 86 54 487657 24 55 75 93 9898 9 487658 23 42 59 89 95 97 12 487659 30 22 56 81 94 96 15 487660 2339 70 85 96 98 12 487661 19 39 57 85 95 97 14 487662 22 27 49 85 92 9517 489009 7 18 46 79 97 99 21 489010 25 24 46 79 96 99 17 489013 26 5377 87 99 100 9 489112 2 11 11 0 18 43 n.d. 496346 1 29 53 85 95 99 19496360 25 37 51 82 96 99 14 496386 19 26 57 83 95 99 16 496387 24 48 7892 98 99 9 496388 12 23 57 78 96 99 18 496391 0 9 54 69 94 98 24 4963922 27 47 79 96 98 20 496393 34 24 60 83 96 99 13 496404 17 39 51 78 96 9816 496405 15 18 35 72 94 99 22 496406 14 25 60 88 98 99 16 496407 26 3962 88 97 99 12

Example 4: Dose-Dependent Antisense Inhibition of Human A1AT in HepG2Cells

Gapmers from of the study described in Example 3 were also tested atvarious doses in HepG2 cells. Cells were plated at a density of 20,000cells per well and transfected using electroporation with 0.31 μM, 0.63μM, 1.25 μM, 2.50 μM, 5.00 μM, and 10.00 μM concentrations of antisenseoligonucleotide, as specified in Table 4. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells and A1AT mRNAlevels were measured by quantitative real-time PCR. Human A1AT primerprobe set RTS3320 was used to measure mRNA levels. A1AT mRNA levels wereadjusted according to total RNA content, as measured by RIBOGREEN®.Results are presented as percent inhibition of A1AT, relative tountreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 4. A1 AT mRNA levels were reduced in adose-dependent manner in some of the antisense oligonucleotide treatedcells, ‘n.d.’ indicates that the IC₅₀ for that compound was notcalculated.

TABLE 4 Dose-dependent antisense inhibition of human A1AT in HepG2 cellsISIS 0.63 2.50 10.00 IC₅₀ No 0.31 μM μM 1.25 μM μM 5.00 μM μM (□M)474061 25 21 18 12 22 39 n.d. 487657 37 25 58 71 82 90 1 487658 13 29 5566 77 87 1.4 487659 12 27 38 60 79 84 1.8 487660 55 77 85 91 92 89 <0.3487661 47 63 69 85 88 86 <0.3 487662 36 55 76 81 85 86 0.4 489009 0 9 1431 51 64 5.4 489010 19 21 23 37 46 50 9.8 489013 16 8 50 55 73 82 2489112 26 0 15 14 12 23 n.d. 496346 0 20 39 49 38 48 6.8 496360 10 12 1954 60 66 3.5 496386 50 66 88 96 97 98 <0.3 496387 52 72 90 96 98 99 <0.3496388 56 67 86 93 97 98 <0.3 496391 17 29 56 77 87 95 1.2 496392 10 3258 77 91 94 1.2 496393 34 22 22 43 50 61 5.7 496404 22 60 82 90 95 970.5 496405 33 37 67 80 92 96 0.7 496406 40 57 80 90 95 98 0.4 496407 5150 77 88 94 98 0.3

Example 5: Dose-Dependent Antisense Inhibition of Human A1AT in Hep3BCells

Gapmers selected from of the studies described in Examples 3 and 4 werealso tested at various doses in Hep3B cells. Cells were plated at adensity of 20,000 cells per well and transfected using electroporationwith 0.31 μM, 0.63 μM, 1.25 μM, 2.50 μM, 5.00 μM, and 10.00 μMconcentrations of antisense oligonucleotide, as specified in Table 5.After a treatment period of approximately 16 hours, RNA was isolatedfrom the cells and A1 AT mRNA levels were measured by quantitativereal-time PCR. Human A1AT primer probe set RTS3320 was used to measuremRNA levels. A1AT mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of A1AT, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 5. A1 AT mRNA levels were reduced in adose-dependent manner in some of the antisense oligonucleotide treatedcells, ‘n.d.’ indicates that the IC₅₀ for that compound was notcalculated.

TABLE 5 Dose-dependent antisense inhibition of human A1AT in Hep3B cellsISIS 0.63 2.50 10.00 IC₅₀ NO 0.31 μM μM 1.25 μM μM 5.00 μM μM (□M)474061 0 12 52 69 89 93 2.0 487657 15 30 45 53 72 88 2.0 489009 9 9 10 832 56 n.d. 489010 0 0 11 9 36 40 n.d. 489013 50 25 39 51 55 65 n.d.489112 0 0 0 0 2 8 n.d. 496346 0 16 31 23 45 49 n.d. 496360 0 15 10 3238 56 9.0 496387 29 63 79 92 98 99 0.4 496393 0 2 11 15 42 55 8.0 4964069 20 63 72 90 96 1.0 496407 18 16 71 82 95 98 1.0

Example 6: Tolerability of Antisense Oligonucleotides Targeting HumanA1AT in CD1 Mice

CD1® mice (Charles River, Mass.) are a multipurpose model of micefrequently utilized for testing safety and efficacy. The mice weretreated with ISIS antisense oligonucleotides selected from the studiesdescribed above, and evaluated for changes in the levels of variousmarkers.

Treatment

Six to seven-week old male CD1 mice were maintained at a 12-hourlight/dark cycle and fed Purina mouse chow 5001 ad libitum. The micewere acclimated for at least 7 days in the research facility beforeinitiation of the experiment. Groups of four CD1 mice each were injectedsubcutaneously twice a week for 6 weeks with 50 mg/kg of ISIS 474061,ISIS 487657, ISIS 487658, ISIS 487659, ISIS 487660, ISIS 487661, ISIS487662, ISIS 487663, ISIS 487664, and ISIS 489013. A group of four CD1mice were injected subcutaneously twice a week for 6 weeks with PBS andserved as the control group. Three days after the last dose at each timepoint, mice were euthanized and organs and plasma were harvested forfurther analysis.

Body and Organ Weights

To evaluate the effect of ISIS oligonucleotides on body and organweights, body weight and liver, spleen, and kidney weights were measuredat the end of the study. The body weights at the end of the study werecompared with body weight at pre-dose. The organ weights of the micetreated with antisense oligonucleotides were compared with thecorresponding organ weights of the PBS control. The results arepresented in Tables 6 and 7. Treatment with ISIS oligonucleotides didnot cause any changes outside the expected range.

TABLE 6 Fold body weight change of CD1 mice compared to pre-dose weightsBody weight change PBS 1.32 ISIS 474061 1.31 ISIS 487657 1.26 ISIS487658 1.29 ISIS 487659 1.30 ISIS 487660 1.37 ISIS 487661 1.39 ISIS487662 1.35 ISIS 487663 1.28 ISIS 487664 1.42 ISIS 489013 1.34

TABLE 7 Fold organ weight change of CD1 mice compared to the PBS controlISIS No Liver Kidney Spleen 474061 1.1 0.9 1.1 487657 1.4 1.0 2.2 4876581.1 1.0 1.5 487659 1.1 1.0 1.5 487660 1.2 1.0 1.7 487661 1.3 0.9 1.6487662 1.3 1.0 1.1 487663 1.0 1.0 1.0 487664 1.3 0.9 1.3 489013 1.2 1.01.5

Plasma Chemistry

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases were measured using anautomated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville,N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured at the time of sacrifice, and the resultsare presented in Table 8 in IU/L. Plasma levels of total bilirubin,creatinine, BUN, and albumin were also measured using the same clinicalchemistry analyzer and are presented in Table 9.

Mice treated with all oligonucleotides except 487664 did not demonstrateany changes in plasma markers outside the expected range.

TABLE 8 ALT and AST levels (IU/L) of CD1 mice ALT AST PBS 44 59 ISIS474061 146 142 ISIS 487657 172 242 ISIS 487658 90 139 ISIS 487659 91 97ISIS 487660 124 97 ISIS 487661 259 182 ISIS 487662 221 143 ISIS 48766353 61 ISIS 487664 508 279 ISIS 489013 79 97

TABLE 9 Plasma bilirubin, creatinine, BUN, and albumin levels of CD1mice Bilirubin Creatinine BUN Albumin (mg/dL) (mg/dL) (mg/dL) (g/dL) PBS0.12 0.13 26.5 2.9 ISIS 474061 0.15 0.12 27.7 2.9 ISIS 487657 0.15 0.1124.2 3.0 ISIS 487658 0.14 0.12 28.0 2.9 ISIS 487659 0.15 0.13 27.1 2.9ISIS 487660 0.14 0.12 25.4 2.9 ISIS 487661 0.12 0.14 29.4 2.8 ISIS487662 0.11 0.12 24.8 2.8 ISIS 487663 0.18 0.11 28.1 3.0 ISIS 4876640.16 0.11 26.0 2.7 ISIS 489013 0.13 0.13 27.2 2.8

Example 7: Efficacy and Tolerability of Antisense OligonucleotidesTargeting Human A1AT in Transgenic PiZ Mice

Transgenic PiZ mice were originally generated by Sifers et al (Nucl.Acids Res. 15: 1459-1457, 1987) by introducing a 14.4 kb DNA fragmentcontaining the entire A1AT gene plus 2 kb of 5′ and 3′ flanking genomicDNA sequences into the germline. The mice were treated with ISISantisense oligonucleotides selected from the studies described above,and the efficacy and tolerability of the antisense oligonucleotides wasevaluated.

Treatment

Five to six-week old male and female PiZ mice were maintained at a12-hour light/dark cycle and fed Purina mouse chow 5001 ad libitum. Themice were acclimated for at least 7 days in the research facility beforeinitiation of the experiment. Groups of four PiZ mice each, consistingof two males and two females, were injected subcutaneously twice a weekfor 4 weeks with 25 mg/kg (50 mg/kg/week) of ISIS 474061, ISIS 487657,ISIS 487658, ISIS 487659, ISIS 487660, ISIS 487661, ISIS 487662, ISIS487663, and ISIS 489013. One group of mice was injected subcutaneouslytwice a week for 4 weeks with 25 mg/kg (50 mg/kg/week) of controloligonucleotide, ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, 5-10-5 MOE gapmerwith no known murine target, SEQ ID NO: 43). One group of mice wasinjected subcutaneously twice a week for 4 weeks with PBS and served asthe control group. Blood samples were collected via tail snip prior todosing and at week 2 and week 4 after dosing. Two days after the lastdose, mice were euthanized and organs and plasma were harvested forfurther analysis.

RNA Analysis

At the end of the study, RNA was extracted from liver tissue forreal-time PCR analysis of human A1AT levels using primer probe setRTS3320. Results are presented as percent inhibition of A1AT, relativeto PBS control, normalized to the house-keeping gene, Cyclophilin. Asshown in Table 10, treatment with some of the ISIS oligonucleotidesreduced A1AT mRNA levels. Specifically, treatment with ISIS 487660reduced A1AT mRNA expression levels. Treatment with the controloligonucleotide, ISIS 141923, did not affect A1AT levels, as expected.

TABLE 10 Percent inhibition of human A1AT mRNA relative to the PBScontrol % ISIS No inhibition 474061 36 487657 29 487658 18 487659 20487660 76 487661 52 487662 53 487663 23 489013 13 141923  6

Protein Analysis

Plasma levels of A1AT were measured with an ELISA kit (Alpco A1AT kit,#30-6752). Results are presented as percent inhibition of A1AT, relativeto the PBS control. As shown in Table 11, treatment with some of theISIS oligonucleotides reduced A1 AT plasma levels. Specifically,treatment with ISIS 487660 reduced A1AT levels. Treatment with thecontrol oligonucleotide, ISIS 141923, did not affect A1AT levels, asexpected.

TABLE 11 Percent inhibition in human A1AT plasma levels relative to thePBS control ISIS No. week 2 week 4 474061 12 18 487657 14 18 487658 1017 487659 12 16 487660 49 61 487661 35 41 487662 32 50 487663 25 41489013 14 29

Example 8: Tolerability of Antisense Oligonucleotides Targeting HumanA1AT in CD1 Mice

CD1® mice were treated with ISIS antisense oligonucleotides selectedfrom the studies described above, and evaluated for changes in thelevels of various markers.

Treatment

Six to seven-week old male CD1 mice were maintained at a 12-hourlight/dark cycle and fed Purina mouse chow 5001 ad libitum. The micewere acclimated for at least 7 days in the research facility beforeinitiation of the experiment. Groups of four CD1 mice each were injectedsubcutaneously twice a week for 6 weeks with 50 mg/kg (100 mg/kg/week)of ISIS 489009, ISIS 489010, ISIS 496346, ISIS 496360, ISIS 496386, ISIS496387, ISIS 496388, ISIS 496391, ISIS 493692, ISIS 496393, ISIS 496404,ISIS 496405, ISIS 496406, and ISIS 496407. Blood samples were collectedvia tail snip prior to dosing and at weeks 2, 3, and 4 after dosing.Three days after the last dose at each time point, mice were euthanizedand organs and plasma were harvested for further analysis.

Plasma Chemistry

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, bilirubin, BUN, albumin, andcreatinine were measured using an automated clinical chemistry analyzer(Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels were measured atthe time of sacrifice, and the results are presented in Table 12.

Mice treated with all oligonucleotides except 489009 and ISIS 496388 didnot demonstrate any changes in plasma chemistry markers outside theexpected range and, therefore, met tolerability requirements.Specifically, treatment with ISIS 496407 was deemed tolerable.

TABLE 12 Levels of plasma chemistry markers of CD1 mice Albu- Creat- ALTAST Bilirubin BUN min inine (IU/L) (IU/L) (mg/dL) (mg/dL) (g/dL) (mg/dL)PBS 48 65 0.15 22.6 2.9 0.17 ISIS 489009 695 412 0.19 23.8 2.8 0.16 ISIS489010 166 225 0.19 21.1 2.9 0.15 ISIS 496346 131 111 0.17 25.4 2.8 0.18ISIS 496360 55 71 0.15 25.5 3.0 0.16 ISIS 496386 53 79 0.16 21.3 2.90.17 ISIS 496387 84 134 0.12 22.4 2.7 0.18 ISIS 496388 528 419 0.11 23.62.4 0.16 ISIS 496391 107 149 0.14 22.4 2.8 0.16 ISIS 496392 64 116 0.1124.3 2.6 0.13 ISIS 496393 130 115 0.10 26.6 3.0 0.17 ISIS 496404 74 910.18 21.6 3.0 0.13 ISIS 496405 79 103 0.12 23.6 2.8 0.14 ISIS 496406 8199 0.14 21.3 2.8 0.15 ISIS 496407 71 102 0.19 18.2 2.7 0.14

Example 9: Efficacy and Tolerability of Antisense OligonucleotidesTargeting Human A1AT in PiZ Mice

PiZ mice were treated with ISIS antisense oligonucleotides selected fromthe studies described above and evaluated for changes in the levels ofvarious markers.

Treatment

Female and male PiZ mice were maintained at a 12-hour light/dark cycleand fed Purina mouse chow 5001 ad libitum. The mice were acclimated forat least 7 days in the research facility before initiation of theexperiment. Groups of four PiZ mice each were injected subcutaneouslytwice a week for 4 weeks with 25 mg/kg (50 mg/kg/week) of ISIS 487660,ISIS 487661, ISIS 487662, ISIS 489010, ISIS 496346, ISIS 496360, ISIS496386, ISIS 496387, ISIS 496391, ISIS 496392, ISIS 496393, ISIS 496404,ISIS 496405, ISIS 496406, and ISIS 496407. A group of four PiZ mice wasinjected subcutaneously twice a week for 4 weeks with PBS and served asthe control group. Blood samples were collected via tail snip prior todosing and at days 12 and 27 after dosing. Three days after the lastdose at each time point, mice were euthanized and organs and plasma wereharvested for further analysis.

RNA Analysis

At the end of the study, RNA was extracted from liver tissue forreal-time PCR analysis of A1AT using human primer probe set RTS3320.Results are presented as percent inhibition of human A1AT, relative toPBS control, normalized to RIBOGREEN®. As shown in Table 13, treatmentwith some of the ISIS oligonucleotides reduced A1AT mRNA levels.Specifically, ISIS 487660, ISIS 487662, ISIS 496386, ISIS 496387, ISIS496392, ISIS 496404, and ISIS 496407 reduced A1AT mRNA levels.

TABLE 13 Percent inhibition of human A1AT mRNA relative to the PBScontrol ISIS No % 487660 72 487661 54 487662 75 489010 49 496346 36496360 24 496386 87 496387 86 496391 69 496392 73 496393 49 496404 70496405 49 496406 74 496407 89

Example 10: Tolerability of Antisense Oligonucleotides Targeting HumanA1AT in Sprague-Dawley Rats

Sprague-Dawley rats are a multipurpose model of rats frequently utilizedfor safety and efficacy testing. The rats were treated with ISISantisense oligonucleotides selected from the studies described inExamples 8 and 9, and evaluated for changes in the levels of variousmarkers.

Treatment

Groups of four Sprague-Dawley rats each were injected subcutaneouslytwice a week for 6 weeks with 50 mg/kg (100 mg/kg/week) of ISIS 487660,ISIS 487662, ISIS 496386, ISIS 496387, ISIS 496392, ISIS 496406, andISIS 496407. A group of four Sprague-Dawley rats was injectedsubcutaneously twice a week for 6 weeks with PBS and served as thecontrol group. Three days after the last dose at each time point, therats were euthanized and organs and plasma were harvested for furtheranalysis.

Plasma Chemistry

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases, BUN, albumin, and creatininewere measured using an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.). Plasma levels were measured at the timeof sacrifice, and the results are presented in Table 14.

Rats treated with ISIS oligonucleotides did not demonstrate changes inplasma chemistry markers outside the expected range and, therefore, weredeemed tolerable in this regard.

TABLE 14 Levels of plasma chemistry markers of Sprague-Dawley rats ALTAST BUN Albumin Creatinine (IU/L) (IU/L) (mg/dL) (g/dL) (mg/dL) PBS 5164 20 4.4 0.20 ISIS 487660 42 70 22 3.3 0.28 ISIS 487662 48 104 29 3.00.26 ISIS 496386 43 85 23 3.3 0.29 ISIS 496387 42 74 23 3.0 0.28 ISIS496392 42 78 21 3.3 0.28 ISIS 496406 70 159 24 2.8 0.23 ISIS 496407 4582 21 3.1 0.26

Body and Organ Weights

To evaluate the effect of ISIS oligonucleotides on body and organweights, body weight and liver, spleen, and kidney weights were measuredtwo days before the rats were euthanized and organ weights were measuredat the end of the study. The results are presented in Table 15.Treatment with ISIS oligonucleotides, except ISIS 496406, did not causeany changes outside the expected range and, therefore, were deemedtolerable in this regard.

TABLE 15 Body and organ weights (in grams) of Sprague-Dawley rats Bodyweight Liver Kidney Spleen PBS 473 1.0 1.0 1.0 ISIS 487660 392 1.3 1.14.1 ISIS 487662 376 1.3 1.3 3.7 ISIS 496386 385 1.2 1.1 4.4 ISIS 496387409 1.1 1.1 4.1 ISIS 496392 368 1.2 1.1 3.0 ISIS 496406 340 1.3 1.2 7.0ISIS 496407 377 1.1 1.1 3.9

Example 11: Dose Response of Antisense Oligonucleotides Targeting HumanA1AT in PiZ Mice

PiZ mice were treated with ISIS antisense oligonucleotides selected fromthe studies described above and evaluated for changes in the levels ofvarious markers.

Treatment

Female and male PiZ mice were maintained at a 12-hour light/dark cycleand fed Purina mouse chow 5001 ad libitum. The mice were acclimated forat least 7 days in the research facility before initiation of theexperiment. Groups of four PiZ mice each were injected subcutaneouslytwice a week for 4 weeks with 12.5 mg/kg, 25 mg/kg, or 37.5 mg/kg ofISIS 487660, ISIS 487662, ISIS 489010, ISIS 496386, ISIS 496392, ISIS496393, ISIS 496404, and ISIS 496407 (weekly doses of 25 mg/kg, 50mg/kg, and 75 mg/kg). One group of four PiZ mice was injectedsubcutaneously twice a week for 4 weeks with 37.5 mg/kg (75 mg/kg/week)of ISIS 141923. One group of four PiZ mice was injected subcutaneouslytwice a week for 4 weeks with PBS and served as the control group. Bloodsamples were collected via tail snip prior to dosing and at week 4 afterdosing. Three days after the last dose at each time point, mice wereeuthanized and organs and plasma were harvested for further analysis.

RNA Analysis

At the end of the study, RNA was extracted from liver tissue forreal-time PCR analysis of A1AT using human primer probe set RTS3320(forward sequence GGAGATGCTGCCCAGAAGAC, designated herein as SEQ ID NO:48; reverse sequence GCTGGCGGTATAGGCTGAAG, designated herein as SEQ IDNO: 49; probe sequence ATCAGGATCACCCAACCTTCAACAAGATCA, designated hereinas SEQ ID NO: 50). Results are presented as percent inhibition of humanA1AT, relative to PBS control, normalized to RIBOGREEN®. As shown inTable 16, treatment with ISIS 487660, ISIS 496386, and ISIS 496407reduced A1AT mRNA levels. Treatment with the control oligonucleotide,ISIS 141923, did not affect A1AT mRNA expression, as expected.

TABLE 16 Percent inhibition of human A1AT mRNA relative to the PBScontrol 25 50 75 ISIS No mg/kg mg/kg mg/kg 487660 40 61 58 487662 19 5251 489010 0 4 9 496386 47 71 84 496392 10 26 39 496393 0 0 0 496404 0 327 496407 22 51 76

Protein Analysis

Plasma levels of A1AT were measured at week 4 with an ELISA kit (AlpcoA1AT kit, #30-6752). Results are presented as percent inhibition ofA1AT, relative to pre-dose levels. As shown in Table 17, treatment withmost of the ISIS oligonucleotides reduced A1AT plasma levels.Specifically, treatment with ISIS 487660, ISIS 487662, ISIS 496386, andISIS 496407 reduced A1AT plasma levels. Treatment with the controloligonucleotide, ISIS 141923, did not affect A1AT levels, as expected.

TABLE 17 Percent change in human A1AT plasma levels relative to pre-doselevels 25 50 75 ISIS No mg/kg mg/kg mg/kg 487660 66 76 83 487662 42 6873 489010 24 22 36 496386 67 82 88 496392 28 48 62 496393 12 34 32496404 47 58 56 496407 46 65 88

Example 12: Dose-Dependent Antisense Inhibition of Human A1AT in PiZMouse Primary Hepatocytes

Gapmers from the study described in Example 11 were also tested atvarious doses in PiZ mouse primary hepatocytes. Cells were plated at adensity of 25,000 cells per well and transfected using electroporationwith 0.63 μM, 2.00 μM, 6.32 μM, 20.00 μM, 63.2 μM, and 200.0 μMconcentrations of antisense oligonucleotide, as specified in Table 18.After a treatment period of approximately 16 hours, RNA was isolatedfrom the cells and A1AT mRNA levels were measured by quantitativereal-time PCR. Human A1AT primer probe set RTS3335_MGB (forward sequenceGACCACCGTGAAGGTGCCTAT, designated herein as SEQ ID NO: 51; reversesequence GGACAGCTTCTTACAGTGCTGGAT, designated herein as SEQ ID NO: 52;probe sequence ATGAAGCGTTTAGGCATGTT, designated herein as SEQ ID NO: 53)was used to measure mRNA levels. A1AT mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of A1AT, relative to untreated controlcells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 18. A1AT mRNA levels were reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 18 Dose-dependent antisense inhibition of human A1AT in PiZ mouseprimary hepatocytes ISIS 2.00 20.0 200.0 IC₅₀ No 0.63 μM μM 6.32 μM μM63.2 μM μM (μM) 487660 0 60 87 90 87 93 0.3 487662 0 27 77 86 85 93 0.4489010 0 0 12 41 82 95 2.3 496392 0 22 81 96 95 96 0.4 496393 0 12 62 9196 97 0.5 492404 0 10 76 95 97 97 0.4 492386 0 43 85 96 96 97 0.3 4924070 30 74 96 96 97 0.4

Example 13: Effect of ISIS Antisense Oligonucleotides Targeting HumanA1AT in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotidesselected from studies described above. Antisense oligonucleotideefficacy and tolerability were evaluated. The human antisenseoligonucleotides tested are also cross-reactive with the complement ofthe rhesus genomic sequence NW_001121215.1 truncated from nucleotides7483001 to 7503000 (designated herein as SEQ ID NO: 14). The greater thecomplementarity between the human oligonucleotide and the rhesus monkeysequence, the more likely the human oligonucleotide can cross-react withthe rhesus monkey sequence. The start and stop sites of eacholigonucleotide to SEQ ID NO: 1 and SEQ ID NO: 14 are presented in Table19. “SEQ ID NO: 1 Start Site” indicates the 5′-most nucleotide to whichthe gapmer is targeted in the human sequence. “SEQ ID NO: 1 Stop Site”indicates the 3′-most nucleotide to which the gapmer is targeted in thehuman sequence. “SEQ ID NO: 14 Start Site” indicates the 5′-mostnucleotide to which the gapmer is targeted in the rhesus monkey genesequence. “SEQ ID NO: 14 Stop Site” indicates the 3′-most nucleotide towhich the gapmer is targeted in the rhesus monkey gene sequence.“Mismatches to SEQ ID NO: 14” are the number of mismatches innucleobases the human oligonucleotide has with the rhesus genomicsequence.

TABLE 19Antisense oligonucleotides complementary to SEQ ID NO: 1 and SEQ ID NO: 14SEQ ID SEQ ID SEQ ID SEQ ID NO 1 NO 1 NO 14 NO 14 Mismatches Start StopStart Start to SEQ ISIS SEQ Site Site Site Site ID 14 Sequence No MotifID NO 1575 1594 14921 14940 0 CCAGCTCAACCCTTCTTTAA 487660 5-10-5 38 15771596 14923 14942 0 GACCAGCTCAACCCTTCTTT 487662 5-10-5 40 1565 1584 1491114930 1 CCTTCTTTAATGTCATCCAG 496386 5-10-5 30 1571 1590 14917 14936 0CTCAACCCTTCTTTAATGTC 496392 5-10-5 34 1421 1440 14767 14786 0GGGTTTGTTGAACTTGACCT 496393 5-10-5 23 1561 1580 14907 14926 1CTTTAATGTCATCCAGGGAG 496404 5-10-5 26 1564 1583 14910 14929 1CTTCTTTAATGTCATCCAGG 496407 5-10-5 29

Treatment

Prior to the study, 36 cynomolgus monkeys were kept in quarantine for a5-week period, during which the animals were observed daily for generalhealth. The monkeys were 2-3 years old and weighed between 2 and 5 kg.Groups of four randomly assigned male cynomolgus monkeys each wereinjected subcutaneously with ISIS oligonucleotide or PBS using astainless steel dosing needle and syringe of appropriate size into theintracapsular region and outer thigh of the monkeys. Seven groups weredosed four times a week for the first week (days 1, 3, 5, and 7) asloading doses, and subsequently once a week for weeks 2-12, with 50mg/kg of ISIS 487660, ISIS 487662, ISIS 496386, ISIS 496392, ISIS496393, ISIS 496404, or ISIS 496407. One group was injectedsubcutaneously with 25 mg/kg of ISIS 496407 four times a week for thefirst week (days 1, 3, 5, and 7) as loading doses, and subsequently oncea week for weeks 2-13. A control group of monkeys was injected with PBSsubcutaneously four times a week for the first week (days 1, 3, 5, and7), and subsequently once a week for weeks 2-13.

Hepatic Target Reduction RNA Analysis

On day 86, RNA was extracted from liver tissue for real-time PCRanalysis of A1 AT using primer probe set rhSERPINAl_LTS00903 ((forwardsequence TCTTTAAAGGCAAATGGGAGAGA, designated herein as SEQ ID NO: 54;reverse sequence TGCCTAAACGCCTCATCATG, designated herein as SEQ ID NO:55; probe sequence CCACGTGGACCAGGCGACCA, designated herein as SEQ ID NO:56). Results are presented as percent inhibition of A1AT mRNA, relativeto PBS control, normalized to the house keeping gene Cyclophilin.Similar results were obtained on normalization with RIBOGREEN®. As shownin Table 20, treatment with ISIS antisense oligonucleotides resulted inreduction of A1AT mRNA in comparison to the PBS control.

TABLE 20 Percent Inhibition of A1AT mRNA in the cynomolgus monkey liverrelative to the PBS control Maintenance % inhibition % inhibition Dose(normalized (normalized ISIS No (mg/kg/wk) RIBOGREEN) Cyclophilin)487660 50 83 87 487662 50 54 37 496386 50 51 38 496392 50 63 55 49639350 33 8 496404 50 12 3 496407 50 45 34 496407 25 25 1

Protein Analysis

On day 85, monkeys in all groups were fasted overnight. The next day,approximately 1 mL of blood was collected into tubes containing thepotassium salt of EDTA. The tubes were centrifuged at 3,000 rpm for 10min at room temperature to obtain plasma. The plasma samples from allgroups were assayed in an automated clinical chemistry analyzer (HitachiOlympus AU400e, Melville, N.Y.) to measure A1AT protein levels using anantibody based assay designed by Olympus. As shown in Table 21,treatment with some of the ISIS antisense oligonucleotides resulted inreduction of A1AT protein levels in comparison to pre-dose levels on day−13.

TABLE 21 Percent Inhibition of plasma A1AT protein levels in cynomolgusmonkey relative to the PBS control Maintenance Dose ISIS No (mg/kg/wk) %inhibition 487660 50 83 487662 50 42 496386 50 30 496392 50 49 496393 507 496404 50 5 496407 50 27 496407 25 29

Tolerability Studies Body and Organ Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health ofthe animals, body and organ weights were measured at day 86. Bodyweights were measured and are presented in Table 22, expressed relativeto pre-dose levels on day 1. Organ weights were measured and the data isalso presented in Table 22, expressed relative to the body weight. Bodyand organ weights after treatment with ISIS oligonucleotides were withinthe expected range.

TABLE 22 Body and organ weights in the cynomolgus monkey (expressed inrelative terms) Dose (mg/kg/ Body ISIS No wk) weight Spleen Kidney LiverPBS — 1.05 0.1 0.5 2.2 487660 50 1.07 0.2 0.8 3.0 487662 50 1.04 0.4 0.73.1 496386 50 1.00 0.4 1.8 3.8 496392 50 1.11 0.3 0.6 3.0 496393 50 1.100.3 0.6 3.0 496404 50 1.01 0.3 0.9 3.2 496407 50 1.01 0.3 0.9 3.5 49640725 1.10 0.3 0.7 3.0

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,approximately 1.5 mL of blood samples were collected from all the studygroups. The monkeys were fasted overnight prior to blood collection.Blood was collected for serum separation in tubes without anticoagulant.The tubes were kept at room temperature for a minimum of 90 min and thencentrifuged at 3,000 rpm for 10 min. Levels of various liver functionmarkers were measured using a Toshiba 200FR NEO chemistry analyzer(Toshiba Co., Japan). Plasma levels of ALT and AST were measured and theresults are presented in Table 23, expressed in IU/L. Bilirubin andalbumin were similarly measured and are presented in Table 34. Liverfunction after treatment with ISIS oligonucleotides was within theexpected range.

TABLE 23 Effect of antisense oligonucleotide treatment on liver functionmarkers in cynomolgus monkey plasma Dose (mg/kg/ ALT AST AlbuminBilirubin ISIS No wk) (IU/L) (IU/L) (g/dL) (mg/dL) PBS — 38 50 4.3 0.15487660 50 52 78 4.1 0.13 487662 50 99 82 3.9 0.11 496386 50 71 77 3.30.10 496392 50 85 64 4.0 0.14 496393 50 68 62 4.2 0.16 496404 50 122 1303.8 0.17 496407 50 56 64 3.6 0.13 496407 25 50 50 3.5 0.13

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,blood samples were collected from all the study groups. The monkeys werefasted overnight prior to blood collection. Blood was collected forserum separation in tubes without anticoagulant. The tubes were kept atroom temperature for a minimum of 90 min and then centrifuged at 3,000rpm for 10 min. Levels of BUN and creatinine were measured using aToshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Results arepresented in Table 24, expressed in mg/dL.

Fresh urine from all animals was collected for urine analysis using aclean cage pan on wet ice. The urine samples were analyzed by theToshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan) for theirprotein to creatinine (P/C) ratio. The data is presented in Table 25.

Kidney function after treatment with ISIS oligonucleotides was withinthe expected range.

TABLE 24 Effect of antisense oligonucleotide treatment on plasma BUN andcreatinine levels in cynomolgus monkeys Dose BUN Creatinine ISIS No(mg/kg/wk) (mg/dL) (mg/dL) PBS — 25 0.9 487660 50 26 0.9 487662 50 261.0 496386 50 69 1.5 496392 50 28 1.0 496393 50 24 0.9 496404 50 28 1.0496407 50 42 1.3 496407 25 36 1.1

TABLE 25 Effect of antisense oligonucleotide treatment on P/C ratio inthe urine of cynomolgus monkeys Dose ISIS No (mg/kg/wk) P/C PBS — 0.0487660 50 0.1 487662 50 0.0 496386 50 5.9 496392 50 0.0 496393 50 0.1496404 50 0.4 496407 50 0.1 496407 25 1.6

Hematology

To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys onhematologic parameters, approximately 0.5 mL of blood was collected onday 86 from each of the available study animals in tubes containingK₂-EDTA. The animals were fasted overnight prior to blood collection.Samples were analyzed for red blood cell (RBC) count, white blood cells(WBC) count, individual white blood cell counts, such as that ofmonocytes, neutrophils, lymphocytes, as well as for platelet count,hemoglobin content and hematocrit, using an ADVIA120 hematology analyzer(Bayer, USA). The data is presented in Tables 26 and 27.

Hematologic parameters after treatment with ISIS oligonucleotides werewithin the expected range.

TABLE 26 Effect of antisense oligonucleotide treatment on various bloodcells in cynomolgus monkeys ISIS Dose WBC RBC Platelets NeutrophilsLymphocytes Monocytes No (mg/kg/wk) (×10³/μL) (×10⁶/μL) (×10³/μL) (%)(%) (%) PBS — 13.2 6.1 490.5 30.8 62.9 3.6 487660 50 13.2 5.8 472.8 16.175.9 5.0 487662 50 11.0 5.5 413.0 19.0 65.7 8.5 496386 50 12.4 5.4 269.545.8 44.6 5.5 496392 50 12.9 5.9 339.5 37.4 52.5 5.4 496393 50 12.7 5.8343.5 34.4 59.6 2.7 496404 50 13.8 6.0 445.0 38.1 53.8 4.9 496407 5010.9 5.5 470.5 37.0 54.3 4.0 496407 25 12.8 5.4 489.8 35.9 56.5 4.2

TABLE 27 Effect of antisense oligonucleotide treatment on hematologicparameters in cynomolgus monkeys Dose Hemoglobin Hematocrit ISIS No(mg/kg/wk) (g/dL) (%) PBS — 13.0 41.9 487660 50 12.8 41.2 487662 50 12.139.2 496386 50 11.1 37.1 496392 50 13.4 42.7 496393 50 12.9 40.9 49640450 13.5 42.0 496407 50 12.4 40.6 496407 25 11.7 38.5

Example 14: Efficacy of Antisense Oligonucleotides Targeting Human A1ATin Transgenic PiZ Mice

Transgenic PiZ mice were treated with ISIS 496407 and its efficacy wasevaluated.

Treatment

Six-week old male and female PiZ mice were maintained at a 12-hourlight/dark cycle and fed Purina mouse chow 5001 ad libitum. The micewere acclimated for at least 7 days in the research facility beforeinitiation of the experiment. One cohort of PiZ mice were injectedsubcutaneously twice a week for 8 weeks with 25 mg/kg (50 mg/kg/week) ofISIS 496407. One cohort of mice was injected subcutaneously twice a weekfor 8 weeks with PBS and served as the control. Two days after the lastdose, mice were euthanized, and organs and plasma were harvested forfurther analysis.

RNA Analysis

At the end of the study, RNA was extracted from liver tissue forreal-time PCR analysis of human A1AT levels using primer probe setRTS3320. Results are presented as percent inhibition of A1AT, relativeto PBS control, normalized to the house-keeping gene, Cyclophilin. Asshown in Table 28, treatment with ISIS 496407 reduced A1AT mRNA levelsin both male and female mice relative to the PBS control.

TABLE 28 Percent inhibition of human A1AT mRNA relative to the PBScontrol % Male 93 Female 81

Protein Analysis

Plasma levels of A1AT were measured once every two weeks with an ELISAkit (Alpco A1AT kit, #30-6752). Results are presented as percentinhibition of A1AT, relative to the values taken pre-dose. As shown inTable 29, treatment with ISIS 496407 reduced A1AT plasma levels.

TABLE 29 Percent inhibition in human A1AT plasma levels relative to thePBS control Week 2 Week 4 Week 6 Week 8 Male 60 80 90 90 Female 70 80 8090

Analysis of Liver A1AT Protein Aggregates

For separation of soluble and insoluble A1AT protein, 10 mg of wholeliver was placed in a buffer consisting of 50 mmol/L Tris HCl (pH 8.0),150 mmol/L KCl, 5 mmol/L MgCl₂, 0.5% Triton X-100, and 80 μL Complete®protease inhibitor stock. The liver tissue was homogenized in apre-chilled Dounce homogenizer with 30 repetitions and then thesuspension was vortexed vigorously. A 1-mL aliquot of the suspension waspassed through a 28-gauge needle 10 times to further homogenize thetissue. The total protein concentration of the aliquot was determined. A5 μg liver sample aliquot was centrifuged at 10,000 g for 30 min at 4°C. The supernatant containing the soluble A1AT fraction was immediatelyremoved into a fresh tube with extreme care being taken to avoiddisturbing the cell pellet, or non-soluble fraction. The cell pelletcontaining the insoluble polymer of A1AT protein was denatured andsolubilized by addition of 10 μL of chilled cell lysis buffer (1% TritonX-100, 0.05% deoxycholate and 10 mmol/L EDTA in PBS), vortexing for 30sec, sonication on ice for 10 min and further vortexing. Both thesoluble A1AT fraction and the solubilized A1AT polymer fraction wereboiled in 2.5× sample buffer (5% sodium dodecyle sulfate, 50% glycerol,0.5 mol/L Tris [pH 6.8], 10% beta-mercaptoethanol, 40% double distilledwater). The samples were then loaded on an SDS-PAGE. Western analysiswas subsequently conducted using goat anti-human alpha-1 antitrypsinNephelometric serum (DiaSorin Inc, Cat #80502). The bands of the Westernblot were quantified using a densitometer and analyzed using ImageJsoftware. The data indicated that both soluble and insoluble A1ATprotein fractions were reduced after treatment with ISIS 496407. Table30 presents the results for the A1AT polymer fraction, expressed aspercentage reduction of the polymer relative to the PBS control.

TABLE 30 Percent inhibition in human A lAT polymer relative to the PBScontrol % Male 32 Female 38

Example 15: Effect of Antisense Inhibition of A1AT in HaltingProgression of A1AT Deficiency Liver Disease in PiZ Mice

The effect of inhibition of A1AT mRNA expression with antisenseoligonucleotides on halting the progression of A1AT deficiency liverdisease was examined in PiZ mice.

Treatment

Male PiZ mice, 6-8 weeks in age, were randomly divided into treatmentgroups of 4 mice each. Three treatment groups were injected with 25mg/kg of ISIS 487660 (SEQ ID NO: 38), administered subcutaneously twicea week for 4, 8, or 12 weeks. Another three groups were injected withPBS, administered subcutaneously twice a week for 4, 8, or 12 weeks. TwoPiZ mice with no treatment administered were included to providebaseline measurements. At the end of each treatment period, the micewere euthanized with isoflurane followed by cervical dislocation. Livertissue was collected and processed for further analysis.

RNA Analysis

RNA isolation was performed using the Invitrogen PureLink™ Total RNAPurification Kit, according to the manufacturer's protocol. RT-PCR wasperformed and A1AT RNA expression was measured using primer probe setRTS3320 and normalized to RIBOGREEN®.

A1AT mRNA expression was assessed in the liver. As shown in Table 31,A1AT mRNA expression in mice treated with ISIS 487660 was inhibitedcompared to the control group in all treatment groups. The mRNAexpression levels are expressed as percent inhibition of expressionlevels compared to that in the PBS control.

TABLE 31 Percent inhibition of AlAT mRNA levels (%) compared to the PBScontrol Weeks of treatment Liver  4 76  8 89 12 80

Protein Analysis

Plasma levels of A1AT were measured once every two weeks with a clinicalanalyzer and Diasorin antibody to A1AT protein. Results are presented aspercent inhibition of A1AT, relative to the values taken pre-dose. Asshown in Table 32, treatment with ISIS 487660 reduced A1AT plasma levelsin all treatment groups.

TABLE 32 Percent inhibition in human A1AT plasma levels relative to thePBS control Weeks of treatment % inhibition  4 63  8 65 12 68

Quantification of A1AT Globules in the Liver

A well-known characteristic of the A1AT deficiency liver disease is thepresence of PAS-positive globules in hepatocytes (Teckman, J. H. et al.,Am. J. Physiol. Gastrointest. Liver Physiol. 2002. 283: G1156-G1165).Analysis was performed on a total tissue area of 2,250,000 μm² usingSpectrum software system (Aperio, Calif.). Hepatocytes were stained withPeriod acid-Schiff stain after diastase treatment of the liver sections.

The average diameters of the globules in each treatment group weremeasured and are presented in Table 33. The total globule area wascalculated and is presented in Table 34. The results indicate thattreatment with ISIS 487660 reduced or halted globule formation inhepatocytes in all treatment groups.

TABLE 33 Average globule diameter (μM) in PiZ mice Weeks of Treatmenttreatment groups Diameter — Baseline 0.42  4 PBS 1.76 ISIS 487660 0.31 8 PBS 2.54 ISIS 487660 0.40 12 PBS 3.33 ISIS 487660 0.48

TABLE 34 Total globule area (μm²) in PiZ mice Weeks of Treatmenttreatment groups Area — Baseline 41,392  4 PBS 95,948 ISIS 487660 58,218 8 PBS 76,074 ISIS 487660 61,260 12 PBS 147,753 ISIS 487660 64,546

Analysis of Liver A1AT Protein Aggregates

For separation of soluble and insoluble A1AT protein, 10 mg of wholeliver was placed in a buffer consisting of 50 mmol/L Tris HCl (pH 8.0),150 mmol/L KCl, 5 mmol/L MgCl₂, 0.5% Triton X-100, and 80 μL Complete®protease inhibitor stock. The liver tissue was homogenized in apre-chilled Dounce homogenizer with 30 repetitions and then thesuspension was vortexed vigorously. A 1-mL aliquot of the suspension waspassed through a 28-gauge needle 10 times to further homogenize thetissue. The total protein concentration of the aliquot was determined. A5 μg liver sample aliquot was centrifuged at 10,000 g for 30 min at 4°C. The supernatant containing the soluble A1AT fraction was immediatelyremoved into a fresh tube with extreme care being taken to avoiddisturbing the cell pellet, or non-soluble fraction. The cell pelletcontaining the insoluble polymer of A1AT protein was denatured andsolubilized by addition of 10 μL of chilled cell lysis buffer (1% TritonX-100, 0.05% deoxycholate and 10 mmol/L EDTA in PBS), vortexing for 30sec, sonication on ice for 10 min and further vortexing. Both thesoluble A1AT fraction and the solubilized A1AT polymer fraction wereboiled in 2.5× sample buffer (5% sodium dodecyle sulfate, 50% glycerol,0.5 mol/L Tris[pH 6.8], 10% beta-mercaptoethanol, 40% double distilledwater). The samples were then loaded on an SDS-PAGE. Western analysiswas subsequently conducted using goat anti-human alpha-1 antitrypsinNephelometric serum (DiaSorin Inc, Cat #80502). The bands of the Westernblot were quantified using a densitometer and analyzed using ImageJsoftware. The data indicated that both soluble and insoluble A1ATprotein fractions were reduced after treatment with ISIS 496407. Table35 presents the results for the A1AT polymer fraction, expressed inarbitrary units.

TABLE 35 Human A1AT monomer and polymer levels Weeks of Treatmenttreatment groups Monomer Polymer  4 PBS 238 2213 ISIS 487660 26 1327  8PBS 675 2598 ISIS 487660 106 1517 12 PBS 159 2317 ISIS 487660 45 1124

Example 16: Effect of Antisense Inhibition of A1AT in Preventing theOnset of A1AT Deficiency Liver Disease in PiZ Mice

The effect of inhibition of A1AT mRNA expression with antisenseoligonucleotides on preventing the onset of A1 AT deficiency liverdisease was examined in PiZ mice.

Treatment

Male and female PiZ mice, 2 weeks in age, were randomly divided intotreatment groups of 4 mice each. One group was injected with 25 mg/kg ofISIS 487660 (SEQ ID NO: 38), administered subcutaneously twice a weekfor 8 weeks (50 mg/kg/week). Another group was injected with PBS,administered subcutaneously twice a week for 8 weeks. Two mice were keptin a separate group and served to establish baseline values ormeasurements of various parameters pre-dose. Two PiZ mice with notreatment administered were included to provide baseline measurements.At the end of each treatment period, the mice were euthanized withisoflurane followed by cervical dislocation. Liver tissue was collectedand processed for further analysis.

RNA Analysis

RNA isolation was performed using the Invitrogen PureLink™ Total RNAPurification Kit, according to the manufacturer's protocol. RT-PCR wasperformed and A1AT RNA expression was measured using primer probe setRTS3320 and normalized to RIBOGREEN®.

A1AT mRNA expression was assessed in the liver. A1AT mRNA expression inmice treated with ISIS 487660 was inhibited by 71% compared to thecontrol group in all treatment groups.

Protein Analysis

Plasma levels of A1AT were measured once every two weeks using aclinical analyzer and Diasorin antibody to A1AT protein. Results arepresented as percent inhibition of A1AT, relative to the values takenpre-dose. As shown in Table 36, treatment with ISIS 487660 reduced A1ATplasma levels.

TABLE 36 Percent inhibition in human A1AT plasma levels relative tobaseline values Week % inhibition 2 40 4 40 6 50 8 40

Quantification of A1AT Globules in the Liver

Hepatocytes were stained with PAS. Analysis was performed on a totaltissue are of 2,250,000 μm² using Spectrum software system (Aperio,Calif.). Hepatocytes were stained with Periodic acid-Schiff stain afterdiastase treatment of the liver sections.

The average diameters of the globules in each treatment group weremeasured and are presented in Table 37. The total globule area in allthe groups was calculated and is presented in Table 38. The resultsindicate that treatment with ISIS 487660 prevented globule formation inhepatocytes in all treatment groups.

TABLE 37 Average globule diameter (μM) in PiZ mice Mouse Treatmentgender groups Diameter Both Baseline 0.23 Male PBS 2.1 ISIS 487660 0.1Female PBS 2.3 ISIS 487660 0.1

TABLE 38 Total globule area (μm²) in PiZ mice Mouse Treatment gendergroups Area Both Baseline 31,856 Male PBS 63,531 ISIS 487660 33,053Female PBS 155,564 ISIS 487660 26,084

Analysis of Liver A1AT Protein Aggregates

For separation of soluble and insoluble A1AT protein, 10 mg of wholeliver was placed in a buffer consisting of 50 mmol/L Tris HCl (pH 8.0),150 mmol/L KCl, 5 mmol/L MgCl₂, 0.5% Triton X-100, and 80 μL Complete®protease inhibitor stock. The liver tissue was homogenized in apre-chilled Dounce homogenizer with 30 repetitions and then thesuspension was vortexed vigorously. A 1-mL aliquot of the suspension waspassed through a 28-gauge needle 10 times to further homogenize thetissue. The total protein concentration of the aliquot was determined. A5 μg liver sample aliquot was centrifuged at 10,000 g for 30 min at 4°C. The supernatant containing the soluble A1AT fraction was immediatelyremoved into a fresh tube with extreme care being taken to avoiddisturbing the cell pellet, or non-soluble fraction. The cell pelletcontaining the insoluble polymer of A1AT protein was denatured andsolubilized by addition of 10 μL of chilled cell lysis buffer (1% TritonX-100, 0.05% deoxycholate and 10 mmol/L EDTA in PBS), vortexing for 30sec, sonication on ice for 10 min and further vortexing. Both thesoluble A1AT fraction and the solubilized A1AT polymer fraction wereboiled in 2.5× sample buffer (5% sodium dodecyle sulfate, 50% glycerol,0.5 mol/L Tris[pH 6.8], 10% beta-mercaptoethanol, 40% double distilledwater). The samples were then loaded on an SDS-PAGE. Western analysiswas subsequently conducted using goat anti-human alpha-1 antitrypsinNephelometric serum (DiaSorin Inc, Cat #80502). The bands of the Westernblot were quantified using a densitometer and analyzed using ImageJsoftware. The data indicated that treatment with ISIS 487660 preventedformation of A1AT protein aggregates in PiZ mice when administered at 2weeks of age.

Example 17: Effect of Antisense Inhibition of A1AT on Liver Fibrosis inthe PiZZ Mice Model

PiZZ mice contain the mutant piZ variant of the human A1AT gene. Themice have accumulation of the mutant human protein in the hepatocytes,similar to that in human patients, causing liver necrosis andinflammation (Carlson, J. A. et al., J. Clin. Invest. 1989. 83:1183-90). The effect of inhibition of A1AT mRNA expression inameliorating liver fibrosis was examined in PiZZ mice.

Study 1 Treatment

Eight week old PiZZ mice were randomly divided into treatment groups of5 mice each. A group of mice was injected with 50 mg/kg/week of ISIS496407, administered subcutaneously for 8 weeks. Another group of micewas injected with PBS, administered subcutaneously for 8 weeks. At theend of each treatment period, the mice were euthanized with isofluranefollowed by cervical dislocation. Liver tissue and plasma was collectedand processed for further analysis.

A1AT mRNA Analysis

RNA isolation from liver tissue was performed using the InvitrogenPureLink™ Total RNA Purification Kit, according to the manufacturer'sprotocol. RT-PCR was performed and A1AT RNA expression was measuredusing primer probe set RTS3320 and normalized to RIBOGREEN®. HepaticA1AT levels were reduced by 91% in mice treated with ISIS 496407compared to the PBS control.

Analysis of Liver Fibrosis Markers

Increased levels of TIMP1 play an important role in the pathogenesis ofliver fibrosis (Arthur, M. J. et al., J. Gastroenterol. Hepatol. 1998.13: S33-8). RNA analysis of TIMP-1 levels was conducted using the primerprobe set mTimp1_LTS00190 (forward sequence TCATGGAAAGCCTCTGTGGAT,designated herein as SEQ ID NO: 57; reverse sequence GCGGCCCGTGATGAGA,designated herein as SEQ ID NO: 58; probe sequenceCCCACAAGTCCCAGAACCGCAGTG, designated herein as SEQ ID NO: 59). Adecrease in TIMP1 levels may lead to a decrease in fibrosis of an organor tissue. TIMP1 levels can be used as a marker for fibrosis in an organor tissue. TIMP1 levels were decreased by 82% in mice treated with ISIS496407.

In addition, analysis of liver damage was conducted by histochemicalstaining. Fibrosis deposition was assessed by Sirius Red staining andquantification of the stain intensity and area. Liver sections from micetreated with ISIS 496407 demonstrated a decrease in staining by 76%(0.20 in arbitrary units vs. 0.83 of the PBS control) compared tostaining of section of the PBS control.

The results indicate that treatment with ISIS 496407 resulted indecreased liver TIMP1 levels and decreased staining of the liver withSirius Red. Hence, antisense inhibition of A1AT resulted in decreasedfibrosis in this mice model.

Plasma Chemistry

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases were measured using anautomated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville,N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured at the time of sacrifice, and the resultsare presented in Tables 39 and 40 in IU/L.

Mice treated with ISIS 496407 decreased transaminase levels compared tothe control, which demonstrated increased levels of both ALT and ASTwith time. The decrease in transaminase levels indicate a prevention oforgan damage, a decrease in organ damage and/or an improvement in organfunction in the oligonucleotide treated mice.

TABLE 39 ALT levels (IU/L) of PiZZ mice Baseline Week 2 Week 4 Week 6Week 8 PBS 57 73 172 153 101 ISIS 59 64 71 64 59 496407

TABLE 40 AST levels (IU/L) of PiZZ mice Baseline Week 2 Week 4 Week 6Week 8 PBS  95 119 234 166 134 ISIS 101  83  90  86  84 496407

Study 2 Treatment

Five week old PiZZ mice were randomly divided into treatment groups of 6mice each. A group of mice was injected with 50 mg/kg/week of ISIS487660, administered subcutaneously for 8 weeks, then 25 mg/kg/week forthree weeks. Another group of mice was injected with PBS, administeredsubcutaneously for 11 weeks. At the end of each treatment period, themice were euthanized with isoflurane followed by cervical dislocation.Liver tissue and plasma was collected and processed for furtheranalysis.

Analysis of Liver Fibrosis Markers

The expression of various genes can be used as markers for fibrosisformation in an organ or tissue. Expression of the following fibrosismarkers in the liver were analyzed: collagen type 1, alpha 1; collagentype IV; collagen type III, alpha 1 (Du, W. D. et al., WorldGastroenterol. 1999. 5: 397-403); MMP12; MMP13; and TIMP1 (Arthur, M. J.Am. J. Physiol. 2000. 279: G245-G249). The results are presented inTable 41. A decrease in the expression of one or more of these fibrosismarkers may be correlative and/or causative of a decrease in fibrosis ofan organ or tissue.

TABLE 41 Inhibition of levels of fibrosis markers in PiZZ mice %inhibition Collagen type 1, alpha 1 75 Collagen type III, alpha 1 57Collagen type IV 21 MMP12 0 MMP13 31 TIMP1 67

In addition, analysis of liver damage was conducted by histochemicalstaining. Fibrosis deposition was assessed by Sirius Red staining andquantification of the stain intensity and area. Liver sections from micetreated with ISIS 487660 demonstrated a decrease in staining by 69%(0.83 in arbitrary units vs. 2.65 of the PBS control) compared tostaining of section of the PBS control. Levels of alpha-smooth muscleactin (SMA), a myofibroblast marker (Hinz, B. et al., Am. J. Pathol.2001. 159: 1009-1020), were also assessed. SMA levels were measured inboth groups. Liver sections from mice treated with ISIS 487660demonstrated a decrease in levels by 40% (0.83 in arbitrary units vs.1.38 of the PBS control) compared to levels in the sections of the PBScontrol.

The results indicate that treatment with an A1AT oligonucleotide, ISIS487660, inhibited A1AT expression leading to a decrease in liverfibrosis as indicated by histochemical staining and the decrease ofmultiple liver fibrosis markers.

Plasma Chemistry

To evaluate the effect of ISIS oligonucleotides on liver and kidneyfunction, plasma levels of transaminases were measured using anautomated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville,N.Y.). Plasma levels of ALT, and AST were measured at the time ofsacrifice, and the results are presented in Tables 42 and 43 in IU/L.

Mice treated with ISIS 487660 decreased liver chemistry marker levelscompared to the control with time. The decrease in transaminase levelsindicate a prevention of organ damage, a decrease in organ damage and/oran improvement in organ function in the oligonucleotide treated mice.

TABLE 42 ALT levels (IU/L) of PiZZ mice Day 1 Day 15 Day 35 Day 56 Day77 PBS 67 56 74 146 78 ISIS 73 34 42  48 58 487660

TABLE 43 AST levels (IU/L) of PiZZ mice Day 1 Day 15 Day 35 Day 56 Day77 PBS 102 105 128 188 106 ISIS  98  56  64  67  69 487660

Example 18: Effect of Antisense Inhibition of A1AT on Reversal ofAggregate Formation in the PiZ Mice Model

The effect of inhibition of A1AT mRNA expression with antisenseoligonucleotides on reversing A1AT aggregate formation was examined inPiZ mice. PiZ mice, at 16 weeks of age, were monitored for 20 weeks forthe effect of antisense inhibition in reversing aggregate formation.

Treatment

Male PiZ mice, 16 weeks in age, were randomly divided into treatmentgroups of 4 mice each. One group was injected with 25 mg/kg of ISIS487660 (SEQ ID NO: 38), administered subcutaneously twice a week for 20weeks (50 mg/kg/week). Another group was injected with PBS, administeredsubcutaneously twice a week for 20 weeks. At the end of each treatmentperiod, the mice were euthanized. Liver tissue was collected andprocessed for further analysis.

Protein Analysis

PiZ mouse livers were homogenized and the soluble and insoluble humanA1AT fractions were separated. Both fractions were separated on sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); equalamounts of total liver protein were loaded per soluble-insoluble pair inquantitative experiments. A1AT protein was detected by a polyclonalantibodies against human A1AT purchased from DiaSorin, Inc. (Stillwater,Minn.) and the secondary antibody was HRP-conjugated rabbit anti-goatantibody (Jackson ImmunoResearch). Western blot was quantitated withImageQuant. Results are presented as percent inhibition of A1AT,relative to the values taken pre-dose at 16 weeks (baseline). As shownin Table 1, treatment with ISIS 487660 reversed insoluble A1AT proteinaccumulation in the liver compared to the PBS control in older mice.

TABLE 44 Percentage of human A1AT liver protein levels relative tobaseline values Levels of soluble Levels of AlAT insoluble A1AT PBS 58107 ISIS 487660 10  65

Analysis of Liver A1AT Protein Aggregates

Histochemical staining for total A1AT protein and Periodic acid-Schiffwith diastase treatment stain (PAS-D, for A1AT aggregates) of 16-weekmice (the age of the mice at the start of the study, which is taken asbaseline), PBS control-treated mice, and ISIS 487660 treated-mice wasperformed. Liver sections from mice treated with ISIS 487660 enhibiteddecreased staining of total A1AT, compared to compared to PBS controland baseline. Liver sections were also stained with PAS-D. PositivePAS-D staining in periportal hepatocytes indicate A1AT accumulation inthe liver, which is associated with the A1AT deficiency disorder.Sections from mice treated with ISIS 487660 exhibited decreased stainingof PAS-D compared to PBS control and baseline.

1. A compound comprising a modified oligonucleotide consisting of 12 to30 linked nucleosides and comprising a nucleobase sequence comprising aportion of at least 8, contiguous nucleobases complementary to an equallength portion of nucleobases 459 to 513, 1349 to 1597, 1561 to 1597,1564 to 1583, 1575 to 1594 of SEQ ID NO: 1, wherein the nucleobasesequence of the modified oligonucleotide is at least 90% complementaryto SEQ ID NO:
 1. 2-9. (canceled)
 10. The compound of claim 1, whereinthe nucleobase sequence of the modified oligonucleotide is at least 95%complementary to SEQ ID NO:
 1. 11-35. (canceled)
 36. A method ofreducing A1AT in an animal comprising administering to the animal amodified oligonucleotide targeting an A1AT nucleic acid sequence asshown in SEQ ID NO:
 1. 37. The method of claim 36, wherein the modifiedoligonucleotide targeting A1AT consists of 12 to 30 linked nucleosidesand is at least 90% complementary to the A1AT nucleic acid.
 38. A methodof treating, ameliorating and/or preventing an A1ATD associated liverdisease in an animal at risk for the A1ATD associated liver diseasecomprising, (a) identifying the animal at risk for developing the A1ATDassociated liver disease; and (b) administering to the at risk animal atherapeutically effective amount of a modified oligonucleotideconsisting of 12 to 30 linked nucleosides, wherein the modifiedoligonucleotide is at least 90% complementary to an A1AT nucleic acid,thereby treating, ameliorating and/or preventing the A1ATD associatedliver disease in the at risk animal. 39-49. (canceled)